materials
Review
Hard Dental Tissues Regeneration—Approaches and Challenges
Mihaela Olaru 1 , Liliana Sachelarie 2, * and Gabriela Calin 2
!"#!$%&'(!
!"#$%&'
Citation: Olaru, M.; Sachelarie, L.;
Calin, G. Hard Dental Tissues
Regeneration—Approaches and
Challenges. Materials 2021, 14, 2558.
https://doi.org/10.3390/
ma14102558
Academic Editor: Ruggero Rodriguez
Received: 30 March 2021
Accepted: 13 May 2021
Published: 14 May 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Materials 2021, 14, 2558. https://doi.org/10.3390/ma14102558
1 “Petru Poni” Institute of Macromolecular Chemistry, 41 A Grigore Ghica Voda Alley, 700487 Iasi, Romania;
olaruma@icmpp.ro
2 Faculty of Medical Dentistry, “Apollonia” University of Iasi, 2 Muzicii Str., 700399 Iasi, Romania;
m_gabriela2004@yahoo.com
* Correspondence: lisachero@yahoo.com
Abstract: With the development of the modern concept of tissue engineering approach and the
discovery of the potential of stem cells in dentistry, the regeneration of hard dental tissues has
become a reality and a priority of modern dentistry. The present review reports the recent advances
on stem-cell based regeneration strategies for hard dental tissues and analyze the feasibility of stem
cells and of growth factors in scaffolds-based or scaffold-free approaches in inducing the regeneration
of either the whole tooth or only of its component structures.
Keywords: stem cells; growth factors; biomaterials; hard dental tissues; scaffold-based and scaffold-
free approach; tooth regeneration
1. Introduction
The tooth consists of three types of highly mineralized tissues, i.e., enamel, dentin, and
cementum. Although the tooth, as a whole, is a mineralized hard tissue similar to bones,
is characterized by a different developmental mechanism. Enamel is a nanocomposite
with intricate hierarchical organization comprising 95 wt.% carbonated hydroxyapatite
(in mature enamel), 4 wt.% water, and 1 wt.% soft organic matrix [1]. Dentin, the main
component of the human tooth, has a similar biochemical composition with bones (70%
hydroxyapatite, 18% collagen, 10% body fluid, and 2% non-collagenous proteins in weight
volume). As follows, the demineralized dentin matrix contains type I collagen, bone mor-
phogenetic proteins, and fibroblasts growth factors [2]. Cementum, the mineralized tissue
that covers the whole root surface, consists of more than 90% type I collagen fibrils, various
types of non-collagenous proteins, i.e., bone sialoprotein, osteopontin, and proteoglycans
and hydroxyapatite [3]. It is well known that the mineralized tissues of the tooth are
characterized by no or limited capacity of self-regeneration [4]. As follows, enamel has
an acellular structure, cementum is characterized by lack of remodeling capacity and/or
limited regrowth in case of a disease-induced resorption, while the regeneration of dentin
is limited and conditioned by the dental pulp stem cell pool, therefore sensitive to any
inflammation or infection process [5].
The continuous increase in the proportion of tooth loss due the action of specific
teeth-adherent bacteria that metabolize sugars into acid and induce the appearance of
dental caries [6] leads to the need to apply for new methods for the tooth regeneration.
Although the conventional restorative materials, such as resin-based composites, porcelain,
and metal crowns proved to be highly effective in preserving hard dental tissues, these
materials are characterized by a rather limited life-span and finally require replacement. In
this context, the development of innovative techniques able to regenerate lost dental hard
tissues can bring significant benefits.
In order to regenerate or initiate the development of a new dental tissue fully inte-
grated within the surrounding medium, the tissue engineering technique relies on the
use of scaffold-based or scaffold free approaches in the presence of suitable stem cells
https://www.mdpi.com/journal/materials
Materials 2021, 14, 2558 2 of 35

and growth factors [7–9]. The scaffold-based approach involves the use of a scaffold in
which cells can be introduced through in vitro planting or via cell homing. This method
depends on the type of the biomaterials used for scaffolds designing, as well as to their
mechanical and physical properties. Furthermore, this approach eliminates the need for
cells manipulation and isolation, thus improving the clinical success and reducing the
cost of the process. The scaffold free technique aims at inducing the gradual process of
embryonic tooth formation under the action of suitable signals in order to obtain tooth
structures that reproduce natural teeth in size and morphology.
The regeneration process of hard dental tissues aims at the regeneration of the indi-
vidual hard components, i.e., enamel, dentin-in correlation with the pulp and cement, as
well as of the whole tooth. Due to its complexity, the regeneration of the whole tooth is a
rather difficult process involving either biologic, genetic, and bioengineering approaches
and involves the substitution of the lost tooth with a bioengineered functional one, re-
constructed using stem cells [10]. The designing of the bioengineered teeth has to meet
several criteria, i.e., to precisely occlude in the dentition, to afford proprioception and
create suitable contacts with surrounding teeth, to convey the masticatory tasks, and to
restore aesthetics [9]. In order to obtain teeth with programmed morphology, it is highly
important to control the orientation, ordering of epithelial mesenchymal cells layers, and of
their interaction with the extracellular matrix. The preferential distribution of cells within
the matrix can be achieved by creating scaffolds via 3D printing, cell seeding or other
techniques [11].
Although many review articles on tooth engineering approaches have been published in
recent years, only a few have focused on the regeneration of the hard dental tissues [12–14].
While these review articles presented excellent summaries of several aspects related to the
regeneration of the hard dental tissues, did not pay particular attention to the correlation
between the characteristics of biomaterials and/or of stem cells and the efficiency of the
regeneration process. This review is focusing on the recent advances in the exploration of
the potential of stem cells for the regeneration of the hard dental tissues, with a special
focus on regeneration of the whole tooth. The outcome of the progress is discussed in
comparison with the current challenges in this area of research.

2. Elements of the Dental Tissue’s Regeneration Process

2.1. Stem Cells
Cell-based therapy, one of the main approaches in the regenerative medicine, re-
quire a suitable cell source, specific methodologies to induce both cell proliferation and
differentiation, maintenance of cell survival, and removal of undesirable cells [15]. The
undifferentiated stem cells are characterized by clonogenic and self-renewing abilities and
can differentiate into several types of cell lineages during growth and development. Taking
into account their origin, stem cells can be classified into embryonic, adult and induced
pluripotent stem cells [15]. As regards their differentiation potential, stem cells can be
categorized into totipotent (capacity to form all types of cells), pluripotent (potential to give
rise to any type of cells), oligopotent (ability to differentiate into in a limited number of cell
types), multipotent (aptitude to form cells of their origin tissue), and unipotent (capacity
to yield one cell type) [16]. The adult stem cells are multipotent, while embryonic and
induced pluripotent ones are pluripotent. The differentiation process allows stem cells to
acquire specific functions and properties depending on the received signals (from within
the cell) or extrinsic (from outside the cell—either mechanical or chemical). The multipotent
human mesenchymal stem cells (hMSCs) can be isolated from a large variety of tissues,
including bone marrow, dental, nervous and adipose tissues, bone, endometrium, muscle,
blood, umbilical cord, amniotic fluid, as well as Wharton’s jelly [17]. hMSCs possess the
ability to differentiate into mesodermal (chondrocytes, osteocytes, and adipocytes) and
non-mesodermal (ectodermal-neurocytes, and endodermal-pancreocytes, and hepatocytes)
lineages [18]. One of the most consistent and available source of autologous stem cells is
represented by the neural-crest derived dental stem cells (DSCs). DSCs are undifferentiated
 hMSCs possess the ability to differentiate into mesodermal (chondrocytes, osteocytes, and
adipocytes) and non-mesodermal (ectodermal-neurocytes, and endodermal-pancreo-
cytes, and hepatocytes) lineages [18]. One of the most consistent and available source of
autologous stem cells is represented by the neural-crest derived dental stem cells (DSCs).
Materials 2021, 14, 2558 3 of 35
DSCs are undifferentiated cells characterized by a multipotent differentiation potential,
unlimited self-renewal and ability to induce tissue regeneration [19]. Up to date, six cate-
gories of characterized
cells dental stem cells
by a were identified
multipotent in the area potential,
differentiation of tooth regeneration, i.e., dentaland
unlimited self-renewal
pulpability
stemtocells (DPSCs),
induce stem cells contained
tissue regeneration [19]. Up to in human
date, exfoliated
six categories deciduous
of dental teethwere
stem cells
(SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle precursor
identified in the area of tooth regeneration, i.e., dental pulp stem cells (DPSCs), stem cellscells
(DFPCs), stem cells from apical papilla (SCAPs), and gingival fibroblast
contained in human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells stem cells
(GFSCs), respectively
(PDLSCs), [20,21].
dental follicle The schematic
precursor representation
cells (DFPCs), stem cellsoffrom
dental stem
apical cells (SCAPs),
papilla involved and
in tooth regeneration can be observed in Figure 1.
gingival fibroblast stem cells (GFSCs), respectively [20,21]. The schematic representation of
dental stem cells involved in tooth regeneration can be observed in Figure 1.

Figure 1. Schematic representation of the dental stem cells involved in tooth regeneration and the
Figure 1. Schematic
associated representation
tissues of thecells
from which these dental
canstem cells involved in tooth regeneration and the
be isolated.
associated tissues from which these cells can be isolated.
Stem cells proliferation and differentiation are regulated by a combination of intrinsic
(several
Stem cells transcription
proliferation factors expressed byare
and differentiation cells) and extrinsic
regulated (signals provided
by a combination of intrinsicby ex-
tracellular
(several matrix factors
transcription (ECM),expressed
growth factors, and
by cells) neighboring
and cells) mechanisms
extrinsic (signals provided by[22]. extra-ECM,
produced
cellular matrixand (ECM), organized
growthby tissue-resident
factors, cells, provides
and neighboring a 3D microenvironment
cells) mechanisms [22]. ECM, pro- to the
ducedstemandcells and protects
organized them againstcells,
by tissue-resident improper
providesdifferentiation, apoptosis and
a 3D microenvironment cellstem
to the damage
cellswhile directing
and protects the maintenance,
them against improper repair and regeneration
differentiation, of theand
apoptosis tissues
cell [23].
damage Furthermore,
while
ECM the
directing confers to the tissues
maintenance, repairitsandmechanical
regenerationproperties (rigidity,
of the tissues [23].and elasticity),ECM
Furthermore, delivers
bioactive
confers to themolecules, and creates properties
tissues its mechanical an environment thatand
(rigidity, facilitates tissue
elasticity), remodeling
delivers in reply
bioactive
to wound
molecules, andhealing
creates [24].
an environment that facilitates tissue remodeling in reply to
Under certain
wound healing [24]. physiological conditions, stem cells are able to transform into functional
cells belonging to a particularconditions,
Under certain physiological tissue [25]. stem
The process
cells areofable
forming complex into
to transform tissues is con-
func-
nected
tional to either the
cells belonging to capability
a particularoftissue
dental stem
[25]. Thecells to differentiate
process of forminginto several
complex linesisafter
tissues
homingtooreither
connected transplantation
the capability [26]ofordental
the secretion
stem cellsof cytokines and growth
to differentiate factors,
into several which
lines
afterinduce
homing theortissue formation under
transplantation [26] the action
or the of locally
secretion of host cells [27].
cytokines and Three
growth main cell cate-
factors,
whichgories are involved
induce the tissueinformation
the formation
underofthe
dental hard
action of tissues, (i) odontoblasts
locally host (tall columnar
cells [27]. Three main
cell categories are involved in the formation of dental hard tissues, (i) odontoblasts (tall for
cells placed at the edge of the dental pulp) derived from mesenchymal cells responsible
dentincells
columnar development,
placed at (ii) theameloblasts
edge of thederived from epithelial
dental pulp) derived from cells mesenchymal
responsible forcells enamel
production, and (iii) cementoblasts (with the origin in the follicular
responsible for dentin development, (ii) ameloblasts derived from epithelial cells respon- cells that can be found
sibleinfor
theenamel
proximity of a tooth
production, root)
and (iii)responsible
cementoblastsfor cementum development
(with the origin [12].
in the follicular cells
that can be The development
found of dentin
in the proximity of involves deposition
a tooth root) and vascularization
responsible of odontoblasts,
for cementum development
[12].this process being followed by the formation of neurons [28]. In addition to their involve-
ment in the dentin of
The development formation, odontoblasts
dentin involves act asand
deposition both nociceptors (“pain
vascularization receptor”) and
of odontoblasts,
defensive cells [29]. During the generation of tooth enamel, ameloblasts
this process being followed by the formation of neurons [28]. In addition to their involve- move toward its
surface and secrete specific proteins (enameling, amelogenin,
ment in the dentin formation, odontoblasts act as both nociceptors (“pain receptor”) and and ameloblastin) that act
as scaffolds for the generation of enamel rods. Cementoblasts are considered to be the
source of cementum intrinsic fibers, as well as a partial foundation for cementum extrinsic
fibers [3]. As regards the neural-crest derived dental stem cells, SHEDs, and SCAPs are
able to differentiate into odontoblasts, DPSCs and PDLSCs differentiate into osteoblasts,
while PDLSCs differentiate into chondrogenic cells and adipocytes [18]. SHEDs have the
ability to form dentin-like tissues, DPSCs are involved in the regeneration of dentin–pulp
complex, SCAPs can induce the dentin and root regeneration, PDLSCs are connected to the
Materials 2021, 14, 2558 4 of 35

regeneration of periodontal tissues and cementum. Furthermore, DFPCs have the capacity
for the regeneration of dentin and cementum [30].

2.2. Growth Factors

Growth factors are critical proteins involved in the development, maturation, preser-
vation, and repairing processes of dental tissues due to their ability to establish a communi-
cation path amongst cells and tissues [31]. More specifically, these proteins are involved in
cell migration, differentiation, and proliferation, as well as gene expression and association
of functional tissues [32,33]. Regarding the use of stem cells in tissue engineering strate-
gies, it is important to understand the processes through which growth factors control
the “fate” of the dental stem cells [34]. As follows, dentin retains its ability to regener-
ate to some degree throughout adulthood, which is thought to arise from the ability of
dental stem cells to produce specific growth factors [31]. The dental mesenchymal cells
from the tooth pulp are differentiated into odontoblasts that are involved in the dentin
deposition process. Dentin does not possess the ability to regenerate when the pulpal
tissue is lost. In order to eliminate this drawback, the growth factors can be included
in tissue engineering scaffolds to adjust their exposure to stem cells and can trigger the
intracellular signal transduction pathway by binding the target growth factor receptor to an
extracellular domain [33]. Various signaling cascades including fibroblast growth factors
(FGF), bone morphogenetic protein (BMP), Wnt/ -catenin (the pathway that regulates the
cells proliferation and differentiation), transforming growth factor beta(TGF- ) and sonic
hedgehog (Shh) are implicated in the regulation of dentogenesis throughout development
and adulthood [35–37]. The tooth morphogenesis is influenced by signaling centers that
control the tissue interactions, as well as the final shape and size of a single tooth. The
specific functions caused by the activation of these pathways can be detected during the
distinct stages of dental tissue differentiation. Some of these stages are advantageous for
cell stemness and proliferation of Shh and FGF. At the same time, TGF- , BMPs, and Wnt
are involved in the phases of postnatal differentiation and induce polarization, migration,
and calcification [38–40].

2.3. Scaffolds for the Regeneration of Hard Dental Tissues

Biomaterials are of a major importance for the regeneration of dental hard tissues. As
such, these biomaterials can act as templates since they can provide the proper environ-
ment for the adhesion, growth, and proliferation of regenerative cells. At the same time,
these biomaterials can act as delivery platforms that support the delivery of implantable
odontogenic cells able to differentiate towards the expected cell type [41]. Furthermore,
these biomaterials can be utilized as delivery platforms for drugs or growth factors that
can further improve the regenerative potential of dental tissues [42–44]. Normally, the
biomaterials used for tooth regeneration must meet some essential criteria, such as biocom-
patibility, non-toxicity, biodegradability without the release of noxious metabolites, ease of
handling, and ability to promote cells proliferation and differentiation. Other important
criteria include inductive factors, good mechanical and physical stability, reduced immuno-
genicity, enhanced vascularity, proper pore size, volume and shape and interconnected
porosity, and capability of cellular surface adhesion or cellular encapsulation. All these
characteristics support the cells functionality and maintaining of their properties in the
oral cavity environment subjected to mechanical forces during mastication, attendance
of microorganisms, variable pH, and temperature [45]. Moreover, the nature of the used
biomaterials is strongly dependent on the characteristics of the hard dental tissue, either
dentin, enamel, or cementum, which is envisioned to be regenerated.
In order to obtain scaffold materials that allows the stem cells and/or growth factors to
generate desired tissues, two main approaches, i.e., top-down and bottom-up, are generally
used in the tissue engineering of dental tissues [46]. In the top-down approach, the stem
cells are seeded in 3D scaffolds made from either natural porous materials, polymers or
decellularized native extracellular matrix. As regards the bottom-up approach, different
Materials 2021, 14, 2558 5 of 35

methods such as cell printing, cell sheets, microwells or self-assembled hydrogels are using
the stem cell aggregates as building blocks to design the desired tissues [46]. Although
various types of materials have been used for dental tissue engineering approaches, i.e.,
natural and synthetic polymers, ceramics, composites, metals incorporated either inside
porous scaffolds, nanofibers, microparticles, meshes, sponges and/or gels, not all them
were suitable for the dental hard tissue regeneration. Typically, dental scaffolds comprise
polymeric biomaterials, bioactive ceramics or composites [47].

2.3.1. Polymers-Based Scaffolds

The polymers used in the designing of scaffolds for the regeneration of hard dental
tissues can be either natural or synthetic. Among the most efficient natural polymers
used in tooth regeneration one can mention type-I collagen, alginate, fibrin, methacrylated
gelatin, and platelet-rich plasma (PRP), while between the synthetic polymers, poly(lactic-
co-glycolic acid), and poly-"-caprolactone can be distinguished. Table 1 is summarizing the
fabrication methods, forms of delivery, advantages and disadvantages of the main natural
and synthetic polymers used for the regeneration of hard dental tissues.
Materials 2021, 14, 2558 6 of 35

Table 1. The main characteristics of the main natural and synthetic polymers used for the regeneration of hard dental tissues.

Biomaterials Type Fabrication Method Forms of Delivery Advantages Limitations References

Resemblance with extracellular
matrix structure, low cytotoxicity and
Plastic compression of immunogenicity, high
hydrogels, biocompatibility, enzymatic
(3D) scaffolds,
multiple unconfined biodegradability, delivery of bioactive High complexity
Type-I collagen Natural biopolymer nanofibrous membranes, [48–54]
plastic compression, molecules for the regeneration of structure
gels, sponges
microextrusion, mineralized tissues, stimulation of
electrospinning osteoblasts differentiation, adeptness
and efficiency to form many shapes,
high tensile strength
High biocompatibility, low toxicity,
easy chemical modification, easy
gelling, relatively inert aqueous
medium, easy encapsulation at room
temperature without organic solvents,
high gel porosity with high diffusion
Poor mechanical
rate, suitable substrate for the release
properties, lack of
of encapsulated transforming growth
Freeze drying, cellular interactions,
factor-beta, capacity to control
freeze-casting, uncontrollable
Alginate Natural biopolymer Scaffolds, gels porosity by simple coatings, [55–59]
dehydrothermal degradation, sterilization
dissolution and biodegradation under
treatment inducing degradation,
normal physiological conditions, slow
non-degradable in
gelling time after the addition of Ca2+
mammals
divalent cations, osteoconductive
and/or bioactive components
promoting osteogenic differentiation,
calcium deposition, biomineralization
and sustaining the natural
regeneration of mineral matrix
Materials 2021, 14, 2558 7 of 35

Table 1. Cont.

Biomaterials Type Fabrication Method Forms of Delivery Advantages Limitations References

3D scaffolds, injectable Appropriate environment for
hydrogels, beads or angiogenesis, formability to 3D
Electrospinning, inkjet microbeads structures, injectability, transforming Weak mechanical
printing, magnetically encapsulating stem cells, of growth factor-beta, controlling properties, fast
Fibrin matrices Natural biopolymer [58,60–72]
influenced self-assembly, coating agents, pro-angiogenic growth factors release, degradation, high
oil-stirring mixture nanoparticles, excellent cytocompatibility, shrinkage
nanofibers, microfibers, non-toxicity of the degradation
microspheres products
Low mechanical
strength, inappropriate
Electrospun, blending,
Excellent cellular compatibility, cell for applications where
photopatterning,
Methacrylated Scaffolds, microgel encapsulation at human body superior tunability as
Natural biopolymer photolithography [73–75]
gelatin arrays temperature, promoting cell viability regards cell adhesion,
microfabrication
and proliferation migration and
technique
degradation mediated by
cells are required
Porogen leaching, gas Incomplete solvent
(3D) scaffolds, Biocompatibility, tunable
Poly(lactic-co- foaming, polymer removal upon
membranes, hydrogels, biodegradability, non-toxicity, high
glycolic acid) Synthetic polymer printing, electrospinning, evaporation, lack of [76–96]
sponges, micro- and cell adhesion and proliferation,
(PLGA) combination of these open-pore structure and
nanoparticles appropriate mechanical properties
methods, self-assembly interconnectivity
Materials 2021, 14, 2558 8 of 35

Table 1. Cont.

Biomaterials Type Fabrication Method Forms of Delivery Advantages Limitations References

Non-toxicity, biodegradable, low
Porogen leaching, melting point, good solubility in
electrospun fibers, Scaffolds with adhered organic solvents, high drug
Poly-"-caprolactone Low in vivo degradation,
Synthetic polymer stereolithography, microspheres, porous permeability, ability of mineralized [97–103]
(PCL) hydrophobicity
solvent casting particle networks PCL scaffolds with apatite to promote
leaching the dental pulp cells growth and
differentiation
Minimal evidence of
dentin development in
Ingrowth of vascularized connective
most of the published
Mixture containing One-step centrifugation, tissues from endodontically
Platelet-rich articles, controversial
proteins, i.e., natural two-step centrifugation Scaffold disinfected root canals, evidence of [104,105]
plasma (PRP) results as regards PRP
polymers of amino acids dentin-like tissues when SCAPs were
therapeutic efficacy in
embedded in a PRP scaffold
periodontal regenerative
procedures
Materials 2021, 14, 2558 9 of 35

2.3.2. Bioactive Ceramic Scaffolds

Bioactive calcium phosphate and glass ceramics represent a group of materials exten-
sively used in tissue engineering applications. Once implanted, these ceramic materials
mediate the formation on their surface of a thin mineral layer of hydroxyapatite, the cal-
cium phosphate mineral that can be found in the dental hard tissues [106]. The cells that
come into contact with the hydroxyapatite-coated ceramics will be able to differentiate and
to produce hard mineralized tissues [107].
Among calcium phosphates, hydroxyapatite, biphasic, and tricalcium phosphate repre-
sents a category of materials with the most references aimed at bone regeneration [108,109].
As follows, 3D calcium phosphate porous granules provided conditions for the growth
and odontogenic differentiation of human dental pulp stem cells, osteoconductivity and ca-
pacity of bone attachment to surrounding tissues [110,111]. The addition of ZnO and SiO2
dopants to tricalcium phosphate scaffolds led to the increase of the bioceramics mechanical
strength, as well as of the cellular proliferation [112].
A scaffold based on a mixture of porous hydroxyapatite, -tricalcium phosphate and
polygricolide fibers yielded the formation of new hard tissues after 6 weeks of implantation,
with dentin-like layers on the inner wall and odontoblasts adjacent aligned to the hard
tissues [113].
Bioactive glasses and glass ceramics represent a combination of different types of
oxides, such as SiO2 , CaO, Na2 O, Fe2 O3 , P2 O5 , and MgO [114]. The glass ceramics are
characterized by variable crystallinity (between 30 and 90%), biocompatibility, opacity or
translucency, and resorbability [115]. Although the use of bioactive and ceramic glasses
as scaffolds in tissue regeneration is restricted by their brittleness, high density and poor
mechanical strength, 3D bioactive scaffolds seeded with human dental pulp stromal cells
induced the osteogenic gene expression, and appearance of sporadic calcified tissues [116].

2.3.3. Composite Scaffolds

The advance registered in the obtaining of biomaterials with special and tailored
properties was possible due to the combinations comprising two different types of scaffold
materials like inorganic materials and synthetic polymers [117]. Although each individual
component can present some specific disadvantages which could eliminate their benefits,
the composites often provide a balance between the strong and weak points of each in-
dividual components, thus providing overall improved properties [118]. Some examples
include the combination of polymers with ceramics for dental tissue regeneration or with
bioactive glasses and ceramics to sustain stem cells differentiation and proliferation into
dentin osteogenic cells [119]. Composites comprising fibrin in mixture with synthetic
polymers or other inorganic materials have been used to obtain 3D dental scaffolds with
improved mechanical properties [120,121]. Other examples include composites designed
for dentin formation, such as the ones containing PLGA porous polymers and different
types of ceramics, i.e., hydroxyapatite, tricalcium phosphate, and calcium carbonate hy-
droxyapatite [122], PLA-based scaffolds doped with dicalcium phosphate and calcium
silicate [123], and PCL with biodentine [124].

3. Enamel Regeneration
While the traditional approach involving the use of specific cells, appropriate scaffold
and grow factors succeeded to lead to the formation of several types of organs or tissues [7],
no successful in vivo enamel tissue engineering using stem cells was reported up to date.
The enamel tissue engineering proved to be quite difficult since the ameloblasts, the enamel-
forming cells and the stem cells or the enamel organ are lost when the teeth erupt [125].
More specifically, the encountered problems were mainly related to the difficulties in
obtaining of viable and potential ameloblast cells, to the complexity of post-translational
modifications of proteins enabling nucleation and elongation of enamel crystals, as well as
the specific coordination of ameloblast cells allowing the organization of enamel crystals
into prism-like patterns [126]. However, some notable achievements in terms of in vitro
Materials 2021, 14, 2558 10 of 35

generation of enamel organ primary ameloblast-like cell culture have been obtained [127].
One primary cell culture enabling mesenchymal–epithelial cells interaction during tooth
morphogenesis succeeded to induce the formation of enamel organ primary cell culture
starting from NIH 3T3 mouse embryonic fibroblasts and a 3D collagen sponge-based
scaffold [128–130]. The primary grown enamel organ cells allowed the expression of
ameloblastin and amelogenin tooth-distinctive genes responsible for the suitable tooth
enamel formation [128].
In addition to the protocols established for the obtaining of enamel organ primary cells,
three types of cell lines, i.e., the rat dental HAT-7epithelial cell line, mouse ALC ameloblast-
lineage cell line, and mouse LS8 cell line presented properties similar to ameloblasts [131].
The use of both HAT-7 epithelial cell line and BCPb8 clonal cells responsible for the
formation of a cementum-like tissue allowed the generation of a matrix which mimics the
mesenchymal–epithelial cell interactions during morphogenesis [132]. HAT-7 epithelial
cell line was found to express an increased level of ameloblastin and amelogenin, the main
parameters as concerns the in vitro amelogenesis process [128]. The same epithelial cell
line allowed the monitoring of ion transport from ameloblasts during the formation of
enamel [133]. Furthermore, HAT-7 were used as supporting cell layers for glycosphinolipid
Gb4 in the transformation process of dental epithelial cells into ameloblasts [134]. ALC
was previously reported to arise as a spontaneously immortalized cell line from C57BL/6
J mice, the usual inbred strain of genetically modified laboratory mouse, that grew on a
type I collagen coated cell culture plates in the presence of an epidermal growth factor. In
addition, ALC was found to allow the expression of amelogenin and tuftelin (the protein
involved in the dental enamel mineralization) [135]. The LS8 cell line was found to be
involved in cytokine dynamics and signaling pathways [136]. LS8 and primary enamel
organ epithelial cells cultured on peptide amphiphiles hydrogels enabled the proliferation
and obtaining of higher levels of ameloblastin, amelogenin, and integrin expression, as well
as the delivery of defined signals for enamel formation [137]. LS8 cell proved to exhibit
higher messenger ribonucleic acid levels for the genes that express secretory-stage activities
(amelogenin, ameloblastin, enamel metalloproteinase, and enamel matrix protein), while
ALC cells presented higher messenger ribonucleic acid levels for the genes that express
maturation-stage activities (odontogenic ameloblast associated proteins, and kallikrein
related-peptidase) [138]. Nevertheless, neither of these cell lines was able to induce the
in vitro formation of enamel-like structures [137,139], most likely due to the different
origin, developing stage and differentiation level of cells, as well as the absence of specific
interaction with the extracellular matrix and/or neighboring tissues.
In an attempt to find new solutions within this research area, several sources of
ameloblast stem cells, such as cervical loop cells, induced pluripotent stem cells, epithelial
cell rest of Malassez (periodontal ligament cells that can be found around a tooth) and
keratinocytes [140–144] were used in combination with specific culture media and growth
factors, yielding the designing of more stable ameloblast cell lines for enamel tissue en-
gineering. Shinmura et al. [142] evidenced the ability of epithelial cell rests of Malassez
seeded on collagen sponge-like scaffolds, together with dental pulp cells, to generate an
enamel-dentin-like structure after eight weeks from transplantation.

4. Dentin Regeneration
Most of the tooth in humans and several other mammalian species is made up of
highly mineralized dentin. Dentin regeneration is usually connected to the treatment of the
dentin–pulp complex. Several aspects related to innervation, revascularization, cell–matrix
interactions, incorporation of growth factors, biodegradation control, remineralization, and
contamination control have to be assured in order to adequate fulfill the dentin–pulp regen-
eration [145]. Due to the essential role of pulp vitality in the stability and homeostasis of the
teeth, the main strategies for dentin regeneration are aimed at maintaining the pulp vitality.
Among the strategies used for the regeneration of dentin–pulp complex, one can mention
the controlled delivery of bioactive agents, tailoring of scaffolds’ physical properties and
Materials 2021, 14, 2558 11 of 35

conjugation of the stimuli responsive components [145]. Among the biomaterials used for
dental pulp tissue engineering, one can mention polylactic or polyglycolic acid [146,147],
fibrin [148,149], collagen [150,151], polylactic-co-glycolic acid [147], polyethylene glycol,
or self-assembling peptides [152–154]. While these types of materials are applicable for
dental pulp tissue engineering, little work has been done in the area of dentin mineralized
tissue regeneration.
Although a number of advances have been made in the treatment of inflamed dental
pulps and irreversible pulp in permanent teeth by using Pro Root MTA® [155] or mineral
trioxide aggregate [156,157] biomaterials, many issues still exist as regards the regeneration
of pulp–dentin complex. Within this context, the regeneration of dentin requires the use
of innovative methods and biomaterials. In respect with this topic, different types of
composites and nanobiocomposites containing bioceramics and various polymers were
used. Table 2 is illustrating the main characteristics of the biomaterials and stem cells used
for dentin regeneration, including their advantages and limitations.
Materials 2021, 14, 2558
Biomaterials
Strontium-free and strontium-doped
calcium silicate mesoporous
nanobioactive glass cements
Bioactive glass nanoparticles modified
with boron and containing 3D scaffolds
based on cellulose acetate, oxidized
pullulan and gelatin
Magnesium-based glass ceramic
scaffolds with copper and zinc ions
Porous hydroxyapatite, -tricalcium
phosphate, powdered hydroxyapatite
and polyglycolic acid bioceramics
Collagen/chitosan biomembrane with
calcium-aluminate microparticles
Table 2. The main characteristics of the biomaterials and stem cells used for dentin regeneration.
Cell Types
Dental pulp stem cells
Human dental pulp stem cells
(hDPSCs)
Dental Pulp Stem Cells (DPSCs)
Dental pulp stem cells (DPSCs)
Human dental pulp cells
12 of 35
Advantages Limitations References
High biocompatibility and strong
odontogenic potential for the
nanobiocements containing strontium,
more degradable and more hydroxyapatite
deposition with strontium substitution, - [158]
absence of cytotoxicity, rapid release of
therapeutic ions, clinically appropriate
teeth defect model for dentin pulp
complex regeneration
High porosity, good mechanical strength,
proliferation and differentiation of hDPSCs
into odontoblasts in vitro conditions, - [159]
improved mechanical properties, lack of
cytotoxicity
Attachment and growth of dental pulp
stem cell for bioceramic scaffolds doped
with zinc, formation of a mineralized tissue Cytotoxicity effect of all bioceramic
[160]
for all copper-doped scaffolds and only for scaffolds doped with copper
zinc-doped ones exposed to lower
temperatures
Osteoconductivity for the scaffolds
containing porous hydroxyapatite and
-tricalcium phosphate, biocompatibility,
Brittleness [113]
resemblance with the mineralized tissues,
positive for type I collagen, osteonectin,
and dentin markers
Stimulation of odontoblastic differentiation,
deposition of mineralized matrix, enhanced - [161]
mechanical properties, cytocompatibility
Materials 2021, 14, 2558
Biomaterials
exfoliated deciduous teeth (SHED),
Human treated dentin
(PDLSCs), dental follicle progenitor
cells (DFPCs), stem cells from apical
Collagen sponges with small amounts
of glycogen synthase kinase inhibitors
Poly(lactide-co-glycolide, PLGA),
composite scaffolds containing 50 wt.%
poly(lactide-co-glycolide) combined
with hydroxyapatite, tricalcium
phosphate or calcium carbonate
hydroxyapatite
PolyL-lactic acid (PLLA) scaffolds
13 of 35
Table 2. Cont.
Cell Types Advantages Limitations References
Human dental pulp stem cells
(DPSCs, stem cells from human
Regeneration of dentin-like tissues in
in vivo conditions, differentiation of DPSCs
periodontal ligament stem cells - [162]
into odontoblasts, appropriate mechanical
properties, non-immunogenicity
papilla (SCAP)
Natural formation of dentine via delivery
Resident mesenchymal stem - [163]
of Wnt signalling agonists
Pure PLGA scaffold inhibits DPSCs
Human dental pulp stem cells Generation of dentin- and pulp-like
proliferation, lack of enamel [122]
(DPSCs) structure, high cell affinity
structure for all composite scaffolds
Formation of a microvascular network and
influx of nutrients and oxygen when
Stem cells from human exfoliated
SHEDs were co-implanted with human Low pH locally generated due to the
deciduous teeth (SHED), primary [164]
endothelial cells, SHED differentiation into degradation of PLLA-based scaffold
human endothelial cells
odontoblast-like and blood vessel-forming
cells in vivo conditions
Materials 2021, 14, 2558 14 of 35

Mandakhbayar et al. [158] evaluated the efficiency of strontium-free and strontium-

doped nanobioactive glass cements for the in vitro and in vivo regeneration of pulp–dentin
complex. The nanobiocements, obtained by mixing the bioactive glass nanopowders with
a phosphate-buffered salin, exhibited a fast release of calcium, silicon, and strontium ions,
known for their therapeutic properties in hard tissue regeneration. The in vitro cultures of
stem cells derived from dental pulp were characterized by a very good biocompatibility
and strong odontogenic potential, especially for the nanobiocements containing strontium.
In vivo conditions, nanobiocements with strontium content yielded the formation of a
higher amount of new dentin. Another research study presented the preparing of bioactive
glass nanoparticles modified with boron and containing 3D scaffolds with tubular morphol-
ogy based on cellulose acetate, oxidized pullulan, and gelatin for dentin regeneration [159].
The 3D scaffolds were obtained by means of porogen leaching and thermally induced phase
separation methods. Scaffold surfaces were completely covered with calcium phosphate
deposits after 14 days immersion in a simulated body fluid. Furthermore, the scaffolds
presented a tubular structure with a similar distribution of bioactive glass nanoparticles
throughout the entire scaffolds and a favorable biodegradability during 1 month. The high
porosity and good mechanical strength of the scaffolds afforded the regeneration of dentin.
The analysis of cell cultures of human dental pulp stem cell and bioactive glass nanoparti-
cles modified with boron illustrated the cell’s attachment, proliferation, distribution and
odontogenic differentiation.
Kontonasaki et al. [160] analyzed the potential of magnesium-based glass ceramic
scaffolds comprising copper and zinc ions and seeded with dental pulp stem cells for
dentin regeneration. The bioceramic scaffolds doped with zinc induced the attachment and
growth of dental pulp stem cell, while the ones doped with copper presented a cytotoxic
behaviour. The formation of a mineralized tissue was noticed for all copper-doped scaffolds
regardless the sintering temperature (up to 890 C), while in case of zinc-doped ones was
evidenced only at the samples exposed to lower temperatures.
Tonomura et al. [113] evaluated the influence of scaffolds geometry (granulated pow-
der of 3D block) of several types of bioceramics, i.e., porous hydroxyapatite, -tricalcium
phosphate, powdered hydroxyapatite, and polyglycolic acid seeded with dental pulp cells
on dentin regeneration. The scaffolds containing porous hydroxyapatite and -tricalcium
phosphate promoted the formation of a dentin-like tissue on the inner wall, in which the
odontoblast-like cells were adjacent distributed toward the hard tissue. Moreover, the same
scaffolds were positive for type I collagen and osteonectin, as well as for dentin markers
dentin sialoprotein and bone sialoprotein.
A biomembrane based on a collagen/chitosan matrix comprising calcium-aluminate
microparticles and simulating dentin composition was used to induce the differentiation of
the human dental pulp cells into odontoblasts. This process was followed by the expression
of odontoblastic phenotypes and deposition of a high amount of mineralized matrix [161].
The biomembrane was obtain by mixing a chitosan solution with a collagen gel in a 1:2
molar ratio, the mixture being further combined with bioactive calcium-aluminate cement.
Culturing of human dental pulp stem cells onto human treated dentin proved to
represent a technique able to regenerate the dentin-like tissues [162]. The dentin specimens
obtained from human third molars were treated with citric acid and ethylene diamine
tetra-acetic acid in order to remove the smear layer. The dentin-like tissues expressed
particular dentin markers such as dentin matrix protein 1 and dentin sialophosphoprotein
after in vivo combination with human treated dentin. Moreover, the cells from the new
dentin-like tissues expressed characteristic human mitochondria antibodies.
Biodegradable collagen sponges were used to deliver small amounts of glycogen
synthase kinase inhibitors that acted as Wnt antagonists able to induce the natural processes
of dentine formation [163]. During collagen sponge degradation over time, the newly
formed dentine was found to replace the degraded sponge, thus leading to a complete
natural tooth repair. Activation of Wnt/ -catenin signaling as an early response to dentin
damage has provided a way to improve natural repair.
Materials 2021, 14, 2558 15 of 35

Several types of 3D scaffolds, including pure poly(lactide-co-glycolide) and other com-

posite scaffolds containing 50 wt.% poly(lactide-co-glycolide) combined with hydroxyap-
atite, tricalcium phosphate, or calcium carbonate hydroxyapatite were evaluated for dentin
regeneration [122]. These scaffolds were obtained through a combination between particle
leaching and phase separation methods. While the scaffolds containing calcium phosphate
were able to fully support the tooth tissue regeneration, the poly(lactide-co-glycolide)-
tricalcium phosphate scaffolds succeeded to induce the proliferation and differentiation of
human dental pulp stem cells and, thus, were more appropriate for dentin formation.
Cordeiro et al. [164] analyzed the morphological characteristics of the formed tissue
when the stem cells derived from human exfoliated deciduous teeth and seeded in polygly-
colic acid scaffolds were transplanted in immunodeficient mice. In the cell-seeded tissues,
the cells from the periphery presented features of active dentin-secreting odontoblasts,
together with a high expression of dentin sialoprotein. The newly formed tissue yielded
cellularity and architecture closely resembling a physiologic dental pulp.

5. Cementum Regeneration
Current research on cementum regeneration has been focused on using stem cells in
combination with suitable scaffolds and growth factors in tandem with different types of
transplantation techniques [12]. The cementum tissue engineering is performing through
the same stem cells therapies applied for the regeneration of the periodontal tissues, i.e.,
(i) cell-based therapy including non- and odontogenic bone marrow (MSCs) and periapi-
cal follicular stem cells (PAFSCs) and (ii) material-based therapy including biomaterial
scaffolds and growth factors [165].
Although adult stem bone marrow mesenchymal (BM-MSCs) cells are able to differ-
entiate into periodontal fibroblasts and to be involved in the regeneration of periodontal
tissues [166], their use in clinical research is rather restricted due to their limited availability.
Periodontal-derived ligament cells (PDLCs) have the capacity to differentiate into cemen-
toblasts and osteoblasts and to form cementum-like tissues [167]. Periapical follicular
stem cells (PAFSCs) are considered one the most promising candidates for cementum
regeneration due to their ability to generate cementum-like matrix [168]. Nevertheless, the
obtaining of these types of cells is challenging and requires tooth extraction. Cementum-
derived cells (CDC) were also used in the periodontal regeneration due to their capacity to
induce periodontal regeneration, being positive for some cementum-specific proteins-like
cementum protein-1 and cementum attachment protein, osteocalcin, amelogenin, and
ameloblastin [169].
Several cementum-specific proteins, including cementum attachment protein, cementum-
derived growth factor and cementum protein-1 were found to induce the formation of a
new cementum [170]. The action of cementum-specific proteins resulted in the inducing
of some signaling pathways connected with mitogenesis, increasing of cytosolic Ca2+
concentration, activating of protein kinase C cascade, and migration and favored adhesion
of progenitor cells. Furthermore, the differentiation into cementoblasts and osteoblasts
allowed the development of a mineralized extracellular matrix similar to cementum. The
addition of cementum protein-1 to a 3D culture of periodontal ligament cells (PDLCs) led
to the increase by twofold of the alkaline phosphatase-specific activity and determined
the expression of cementogenic markers, as well as the development of new types of
cementum-like tissues [171].
The stem cells from periodontal ligament (PDL) fibers, alveolar bone and gingiva
were used as sources for cementoblasts and yielded the appearance of cementum-like
mineralized nodules and cementum-specific markers [172]. Periodontal ligament stem
cells (PDLSCs), adipose tissue-deprived stem cells (ADSCs) and the stem cells from the
dental follicle (DFSCs) were capable to differentiate toward cementoblasts and to form
a cementum-like tissue [173]. The histological examination of a treated dentin matrix
combined with dental follicle cells (DFCs) and implanted subcutaneously in a mice dor-
sum revealed the swirling alignment of DFCs in several layers positive for fibronectin,
Materials 2021, 14, 2558 16 of 35

integrin 1, collagenase I and alkaline phosphatase and induced the development of a new
cementum–periodontal complex [174]. The transplantation of such types of stem cells at
the place of periodontal defects can represent a reliable technique for the regeneration
of cementum.
Three categories of transplantation techniques were utilized for cementum regener-
ation, i.e., transplantation of a scaffold with or without cell content, cellular pellet trans-
plantation, and injection of stem cells [166]. Transplantation of a scaffold may increase the
efficiency of cementum regeneration since this scaffold can decompose in certain conditions
and may allow the cells to remain at the injury site. However, this efficiency depends on
the compatibility between the scaffold and stem cells.
Cementum, along with dentin and enamel, is a dental tissue belonging to periodon-
tium, with particular functional properties strongly connected to its hierarchical structure.
As a consequence, the successful regeneration of cementum remains a challenging since
it requires a succession of coordinated responses throughout the various hard and soft
tissue interfaces [175]. Tissue engineered scaffolds can provide a proper microenvironment
for cementum regeneration because they can promote cells recruitment, optimization of
cell-based treatments and control the release of growth or gene factors or proteins.
Recent advances on scaffold constructions for cementum regeneration include multi-
phasic and 3D printed scaffolds and hydrogels. Although the therapies with or without
cells, scaffolds, and growth factors represent different types of regenerative treatments,
these strategies are frequently employed in combination with each other.

5.1. Multiphase Scaffolds for Cementum Regeneration

Multiphase scaffolds are designed to resemble the structural organization and biomimetic
functions of the native tissues and are characterized by variations of their architectural
characteristics (porosity, pore associations) and biochemical compositions along the entire
construct [176]. Taking into account the interactions between various hard and soft tissues
and the complex structure of periodontium, Ivanovski et al. [175] reported the main
parameters of a multiphasic scaffold for the tissue engineering of periodontum, i.e., (i)
formation of compartmentalized bone and periodontal tissue that integrates over time,
(ii) sustaining of cementum formation on root surface, and (iii) development of suitably
oriented ligament fibers that interpolate into cementum and newly formed bone.
Cell sheet technology proved to represent a valuable, clinically applicable approach
for cementum regeneration. The efficacy and safety of the allogeneic transplantation of
periodontal ligament and odontogenic bone marrow cell sheets using a horizontal canine
periodontal defect model was reported [175]. The periodontal ligament and odontogenic
bone marrow cell sheets were cultured on thermoresponsive dishes, while a gel comprising
a mixture of collagen and -tricalcium phosphate was placed at the bone defects. The
allogeneic transplantation group presented a significantly higher regeneration of the new
formed cementum as compared with the autologous ones.
Several authors proposed a strategy in which various types of periodontal cell sheets
applied on the root surface induced the formation of a new cementum, as well as the
promotion of periodontal attachment [177–183]. This approach implied the use of a
thermoresponsive cell culture dish to harvest the cells without injury the extracellular
matrix. The implantation of the fragile tissues was performed by using different non-
or biodegradable membranes, such as hyaluronic acid [177], fibrin gel [178], poly(N-
isopropylacrylamide) [179], polyglycolic acid [181], trypsin/ethylenediaminetetraacetic
acid, collagenase/dispase [182], and non-absorbable GoreTex membrane [183] to enable the
handling and settlement of the cells. Although promising, this strategy proved to depend
on the insufficient biomechanical stability of the membranes or cell sheet constructs.
Vaquette et al. [184] reported the obtaining of a biphasic scaffold based on poly-
caprolactone containing -tricalcium phosphate comprising a bone compartment and an
electrospun membrane. In order to achieve the periodontal regeneration, multiple peri-
odontal ligament (PDL) cell sheets and/or osteoblasts were included in the electrospun
Materials 2021, 14, 2558 17 of 35

membrane. After implantation of the biphasic scaffolds seeded with cell for eight weeks
in a dentin block from a subcutaneous model of stability of the cell sheets on the dentine
surface of the athymic rat, the appearance of a thin cementum-like tissue was observed on
dentin surface in case of the scaffolds containing PDL cell sheets. As follows, around 67%
of the samples holding PDL cell sheets presented cementum-like tissues at the dentin–cell
interface. At the same time, the samples holding PDL cell sheets showed a higher degree
of cementum root coverage as compared with the ones without cell sheets, in which only
17% of the groups exhibited cementum formation. Although no vertical insertion of col-
lagen fibrils into the new formed cementum was noticed, the fibrous attachment inside
the newly mineralized tissue throughout almost the entire width of dentin surface was
constantly maintained.
Table 3 is presenting the main characteristics of some of the biomaterials and stem
cells used for cementum regeneration, including their advantages and limitations.
Materials 2021, 14, 2558
Biomaterials
Canine periodontal defect model
filled with collagen and -tricalcium
phosphate mixture
Hyaluronic acid carrier
Poly(N-isopropylacrylamide)
Polyglycolic acid
Trypsin/ethylenediaminetetraacetic
acid, collagenase/dispase
Gore-Tex membrane
Table 3. The characteristics of some of the biomaterials and stem cells used for cementum regeneration.
Type of Cells
Periodontal ligament derived
multipotent mesenchymal stromal
cells (PDL-MSC)
Periodontal ligament containing
stem cells
Human periodontal ligament
(HPDL) cells
Canine periodontal ligament
(PDL)derived cells
Human PDL (hPDL) cells, human
adipose-derived stem cells
(hADSCs), gingival fibroblasts
(hGFs), bone marrow-derived
mesenchymal stem cells
(hBMMSCs)
Periodontal ligament (PDL) cells
18 of 35
Advantages Limitations References
Decrease of cell viability as a consequence
Substantial regeneration of the newly formed
of lentiviral transduction, the size of the
cementum tissue, periodontal tissue [175]
periodontal defect is too large to deliver
regeneration without any side effects
proper nutrients and blood
Formation of a new cementum, regeneration of
periodontal tissues, adherence and A partial regeneration was obtained [176]
proliferation of periodontal ligament cells
Regeneration of periodontal ligament tissues,
enhanced cell proliferation, cell migration and
- [177]
differentiation toward mineralized tissues
upon the addition of ascorbic acid
Ability of osteoblastic differentiation, formation
of a cementum tissue connected with oriented
collagen fibers, appropriate orientation of - [178]
cementum and periodontal ligaments in the
experimental group
Osteogenic potential in vivo and in vitro Low chondrogenic and adipogenic
conditions, promotion of calcium deposition potentials of hPDL cells, lack of calcified [179]
and rapid proliferation of hPDL cells tissues for hPDL cells
Osteogenic differentiation expressing
osteopontin (OPN) and bone sialoprotein (BSP),
appearance of cementum-like tissues in vivo
- [180]
conditions, including PDL fibers and Sharpey’s
fibers, in the presence of an osteogenic
differentiation medium
Materials 2021, 14, 2558
Biomaterials
Fibrin gel
Polycaprolactone with -tricalcium
phosphate
Poly(lactic-co-glycolic acid) (PLGA)
Poly(glycolic acid) (PGA),
polycaprolactone (PCL)
Type of Cells
Multilayered human periodontal
ligament cells
Multiple periodontal ligament
(PDL) cells
Immortalized cementoblasts
(OCCM) transduced with antagonist
of platelet-derived growth factor
(PDGF) signaling (ADGF-1308),
adenovirus encoding PDGF
(PDGF-A), control virus (GFP)
Primary human gingival fibroblast
(hGF) cells
19 of 35
Table 3. Cont.
Advantages Limitations References
Formation of undeveloped cementum-like
tissues and periodontal ligaments resembling The appearance of a cementum–periodontal
Sharpey’s fibers in an osteodifferentiation ligament complex was not observed in all [181]
medium, increased calcium deposition and experimental samples
alkaline phosphatase activity
Increased cell sheets stability on dentine
surface, appearance of a discontinuous Frequent cell monitoring [182]
cementum-like tissue
Formation of well differentiated cementoblasts, Inhibitory effect of PDGF-A on
[183]
cells attachment to PLGA scaffolds cementogenesis
Appearance of a human tooth Lack of symmetric design and adequate
dentin–ligament–bone complex in porcine mechanical properties for hybrid scaffolds [184]
mandibulae with surgically created defects only
Materials 2021, 14, 2558 20 of 35

5.2. 3D Printed Scaffolds for Cementum Regeneration

In order to promote the formation of interfacial tissue amongst tooth dentin and
periodontal ligament fibrous tissues, 3D biopolymeric scaffolds based on different types
of biomaterials have been obtained. Poly(lactic-co-glycolic acid) (PLGA) scaffolds with
open pores played an important role in transporting cementoblasts and cementogenesis-
promoting products such as platelet-derived growth factor-BB for the in vivo activation
of cementogenesis process [185,186]. Cementoblasts were transduced with adenoviruses
encoding either PDGF-A, an antagonist of platelet-derived growth factor (PDGF) or no
treatment. The constant exogeneous PDGF-A delayed the formation of the mineral tissue
under the action of cementoblasts, while PDGF induced the mineral tissue neogenesis [185].
In another study, periodontal ligament fibroblasts, cloned cementoblasts and dental follicle
cell seeded onto 3D PLGA scaffolds were used to study their influence in the promotion of
cementum formation both in vitro and in vivo conditions [186]. The study reported that
only cementoblasts promoted the mineral formation and that a critical mass of completely
matured bone-cementum cells is necessary for periodontal regeneration.
Park et al. [187] used a solid free-form fabrication approach (3D wax printing system)
and a computational topology design for the obtaining of polymeric scaffolds based on
polycaprolactone and poly(glycolic acid) for the in vivo formation of a dentin–ligament–
bone complex. The two polymers were selected taking into account their biodegradation
rate for periodontal ligament fibrous tissue and for the formation of mineralized-like tissues,
respectively. Poly(glycolic acid) was positioned at the periodontal ligament interface
and poly-"-caprolactone in the bone architecture. The newly formed tissues yielded the
interfacial production of fibers parallel and oblique oriented within the polymeric scaffolds
and subsequent development of cementum-like tissues in association with fibrous and
vascular structures. It was speculated that the associated fibrous tissues represented the
transition benchmark of the cementogenesis process occurring at the dentin surface, as
well as the early integration of the fibrous bundles into dentin.
Park et al. [188] manufactured a fiber-guiding biomimetic 3D scaffold in order to
promote the formation and integration of ligament, bone and cementum. The fiber-
guiding biomimetic 3D scaffold was obtained by casting poly-"-caprolactone solution
onto cylindrical-shaped periodontal ligament fiber guiding architectures. Cementum-like
tissues were deposited onto the dentin surfaces containing fiber-guiding scaffolds, whereas
the ligament cells randomly oriented induced a lesser amount of cementum. Furthermore,
the results illustrated the correlation between the topography of the designed scaffold and
the functional renewal of regenerated tissues. In a later study, the protocol for the fast
prototyping, manufacturing, surgical implantation, as well as the evaluation of poly-e-
caprolactone fiber-guiding 3D scaffolds for controlling the fiber alignment and enabling
morphogenesis process of bone–ligament complex was established [189]. The shape of the
fiber-guiding 3D scaffold influenced both cell and tissue association, as well as the appear-
ance of type-I collagen bundles (Sharpey’s collagen fibers that serve as liaisons between
the mineralized-like tissues) perpendicularly oriented toward the tooth root surface.
3D multiphase bioscaffolds with periodontal ligament stem, dental pulp stem, and
alveolar bone stem progenitor cells were used for the regeneration of periodontium [190].
The scaffold was obtained through 3D printing of polycaprolactone, whose biocompatible
and biodegradable properties allowed the scaffold manufacturing with controlled pore,
elasticity and tailored shape. In order to obtain the 3D printed scaffold, a mixture of
90:10 wt.% polycaprolactone and hydroxyapatite was used. The scaffold seeded with dental
pulp stem progenitor cells yielded the formation of periodontal ligament-like collagen
fibers inserted inside mineralized matrix containing dentin sialophosphoprotein-positive.
The same situation was encountered in case of the cementum matrix protein 1-positive and
in bone-like tissue comprising bone sialoprotein-positive.
Cho et al. [191] reported the obtaining of growth factor-releasing 3D polycaprolactone
scaffolds to achieve cementum formation. The 3D printed polycaprolactone scaffolds
contained poly(lactic-co-glycolic acids) microspheres, connective tissue growth factors,
Materials 2021, 14, 2558 21 of 35

and bone morphogenetic proteins such as bone morphogenetic protein-2 and bone mor-
phogenetic protein-7. After 6 weeks, all groups containing growth factors induced the
formation of a newly cementum-like layer on the top of dentin.
Several types of topologies of different biodegradable biomaterials were investigated
in order to trigger the canonical Wnt or Wnt/ -catenin signaling (the pathway that reg-
ulates the cells proliferation and differentiation) pathways, processes involved in the
induction of cementogenesis process. Bioceramics composed of hydroxyapatite with 3D
hierarchical structure (micro- and nanorods) prepared via hydrothermal reaction of ↵-
tricalcium phosphate were investigated, among others, for cementogenic differentiation
of the human periodontal ligament stem cells [192]. The results evidenced that these
hydroxyapatite-based bioceramics promoted the expression of osteogenic/cementogenic-
related markers including cementum protein and cementum attachment protein and al-
lowed the gene expression of the principal genes of canonical Wnt signaling, i.e., -catenin
and low-density lipoprotein receptor-related protein 5.

5.3. Gels and Hydrogels for Cementum Regeneration

As opposed to 3D scaffolds, gels and hydrogels injectable materials can easily adopt
the shape of the irregularly bone defects and requires negligible invasive surgical methods.
Gels and hydrogels can achieve the in situ delivery of bioactive molecules or drugs in liquid
forms over an anticipated period of time [193]. Within this context, several delivery poly-
meric systems were developed for drug sustained release. Wang et al. [192] investigated
the regenerative efficiency of two systems, i.e., one based on propylene glycol alginate gel
containing a fibroblast grown factor and one comprising a mixture of propylene glycol
alginate gel, bone morphogenetic protein and a cementum composite on the regeneration of
periodontal defects. This cementum composite was consisting of 59.1 wt.% alpha-tricalcium
phosphate, 1.5 wt.% carboxymethylcellulose and 39.4 wt.% cryo-ground propylene glycol
alginate particles. The experimental group exhibited a substantial improvement in regards
cementum, ligament, and epithelial regeneration as compared to the control one.
The single topical application of gelatin biodegradable hydrogel containing different
amounts of human recombinant fibroblast growth factor was analyzed to study the peri-
odontal regeneration in case of primate models [194,195]. The gelatin hydrogel attained
the sustained release of the human recombinant fibroblast growth factor as a consequence
of the hydrogel degradation. Furthermore, the degradation rate was controlled by varying
the level of gelatin crosslinking. The formation of a new cementum containing Sharpey’s
fibers was noticed on the root surfaces, while the novel connective tissue fibers and peri-
odontal ligament fibers were found to insert into the newly formed bone and cementum.
A similar analysis was performed by applying a gelatin gel carrier containing recombi-
nant basic fibroblast growth factor on beagle dog models [195]. The topical application of
biodegradable gel with the growth factor yielded after 4 weeks the formation of periodontal
ligament together with new cementum and bone. The histological analysis revealed the
fill of the physiologic space between the new formed cementum and bone with organized
collagen fibers.
A tri-layered hydrogel nanocomposite scaffold designed for cementogenic, osteogenic
and fibrogenic differentiation of the human dental follicle stem cells was obtained by
assembling poly(lactic-co-glycolic acid)-chitin/nanobioactive glass ceramic/cementum
protein-1, poly(lactic-co-glycolic acid)-chitin/fibroblast growth factor 2, and poly(lactic-
co-glycolic acid)-chitin/nanobioactive glass ceramic/platelet-rich plasma derived growth
factors, respectively [196]. The histological analysis evidenced the development of a new
cementum with aligned cementoblasts along the root surface, of a new fibrous periodontal
ligament attached to the new cementum, as well as of alveolar bones.

6. Whole Tooth Engineering

One of the biggest challenges of dental tissue engineering is related to the obtain-
ing of the complex structure of a whole tooth comprising both soft tissues (dental pulp,
Materials 2021, 14, x FOR PEER REVIEW 22 of 35

6. Whole Tooth Engineering

Materials 2021, 14, 2558 One of the biggest challenges of dental tissue engineering is related to the obtaining 22 of 35
of the complex structure of a whole tooth comprising both soft tissues (dental pulp, ves-
sels, and stroma), and hard tissues (dentin, enamel, and cementum) of a pre-defined mor-
vessels,and
phology and shape
stroma),in and
which hardthetissues (dentin,ofenamel,
interactions and cementum)
the growth of a pre-defined
and transcription factors
morphology and shape in which the interactions of the growth and
(homeobox genes that act as main regulators during embryonic development), as well transcription factors
as
(homeobox genes that act as main regulators during embryonic development),
cytokines direct the micro- (root formation, cusp number) and macromorphological (tooth as well
as cytokines
length, and crowndirect thetooth
size) micro- (root formation,
development cusp number)
with suitable andfunctionality
biological macromorphological
in vivo
(tooth length, and crown size) tooth development with suitable biological
conditions [197]. In order to attain its complete tissue homeostasis and functionality, functionality
the
in vivo conditions [197]. In order to attain its complete tissue homeostasis and functionality,
newly designed tooth must be innervated. The whole tooth engineering technique is
the newly designed tooth must be innervated. The whole tooth engineering technique
based on the reciprocal interactions between the mesenchymal and dissociated embryonic
is based on the reciprocal interactions between the mesenchymal and dissociated embry-
dental epithelial cells mediated by well-regulated signaling pathways such as bone mor-
onic dental epithelial cells mediated by well-regulated signaling pathways such as bone
phogenetic protein (BMP), fibroblast growth factor (FGF), Hedgehog (HH), and Wnt (mer-
morphogenetic protein (BMP), fibroblast growth factor (FGF), Hedgehog (HH), and Wnt
ger of wingless and Int-1) pathways [198]. Since the signaling pathways constitute a sep-
(merger of wingless and Int-1) pathways [198]. Since the signaling pathways constitute a
arate tooth “signaling program”, the bioengineering (regrowth process) of a new tooth is
separate tooth “signaling program”, the bioengineering (regrowth process) of a new tooth
possible by reiterating this signaling program at the scale of the whole tooth.
is possible by reiterating this signaling program at the scale of the whole tooth.
The schematic representation of the components required for the whole tooth regen-
The schematic representation of the components required for the whole tooth regen-
eration, i.e., suitable stem cells, signaling molecules, scaffolds, and homeobox genes is il-
eration, i.e., suitable stem cells, signaling molecules, scaffolds, and homeobox genes is
lustrated in Figure 2.
illustrated in Figure 2.

Figure 2. Summary of stem cell therapy for inducing tooth regeneration (Adapted with permission
Figure 2. Summary of stem cell therapy for inducing tooth regeneration (Adapted with permission
from ref. [199]. Copyright 2018 Elsevier).
from ref. [199]. Copyright 2018 Elsevier).
The generation of a whole tooth was accomplished by several methods, such as “organ
The or
germ” generation of a whole
“bioengineered tooth
organ was methods
germ” accomplished[200],by several methods,
stimulation such as “or-
of the formation of a
gan germ” or “bioengineered organ germ” methods [200], stimulation of the
new tooth (or third dentition) [201], engineering scaffolds of dental tissues [202], gene- formation of
a new tooth
handled (or third
tooth dentition)
regeneration [201],
[203], engineering
chimeric scaffolds
tooth tissue of dental[111,204],
engineering tissues [202], gene-
in situ tooth
handled tooth regeneration [203], chimeric tooth tissue engineering [111,204],
regeneration by stimulating the tooth replacement ability [198] and cell–cell or tissue–cellin situ tooth
regeneration
recombination by stimulating the tooth
via embryonic tooth germ
replacement ability [198]Currently,
cells [200,205–209]. and cell–cell
theor tissue–cell
main research
recombination via embryonic tooth germ cells [200,205–209]. Currently, the
directions for whole tooth regeneration are focusing on the in situ tooth regeneration main researchby
directions for the
stimulating whole tooth
tooth regeneration
replacement are
ability, focusing on the
bioengineered organin germ,
situ tooth regeneration
and tissue by
engineering
stimulating
approaches the tooth replacement ability, bioengineered organ germ, and tissue engineer-
[30,197,198].
ing approaches [30,197,198].
6.1. In Situ Tooth Regeneration by Stimulating the Tooth Replacement Ability
Recent studies evidenced that vertebrate teeth can phylogenetically develop during the
extension of the odontogenic competence of the external dermal denticles [210]. Among all
vertebrates, many amphibians, reptiles, and most of the teethed fishes are polyphyodonts,
i.e., possess the ability to continuous regenerate their teeth throughout their entire lives. In
the case of mammals, several species including human ones are only diphyodont (with the
ability to form a second dentition), while other species such as mouses are monophyodont
Materials 2021, 14, 2558 23 of 35

(develop a single set of teeth during the growth phase) [211]. Although the replacement
capacity of vertebrate teeth has been significantly reduced over the course of evolution [212],
in recent years the emphasis has been on either biological replacement of the dental tissues
or on the in vivo regeneration of the whole tooth by revitalizing its regenerative potential.
The requirement for tooth replacement is the attendance of successional dental lamina
(dental lamina found on the lingual side of a first tooth) that holds the ability to induce
the odontogenesis process. The presence of the rudimentary successional dental lamina,
considered as a possible source for a third dentition, was seldomly identified in case of
humans. Both dental and successional dental lamina were found to be recognized by
Sox2 stem cell marker [213]. The successional dental lamina proved to be triggered by the
increase of the canonical Wnt pathway (the signal transduction pathway group involved
in the cumulation of -catenin inside cytoplasm) and yielded the tooth formation in case
of alligators and snakes [214]. Furthermore, the appearance of a transitory rudimentary
successional dental lamina was observed during mice tooth development, animals that
normally never replace their teeth [213]. The stabilizing of Wnt signaling in rudimentary
successional dental lamina by application of suitable factors or genes is regarded as a
strategy for the future whole tooth regeneration.

6.2. Whole Tooth Regeneration through Bioengineered Organ Germ Method

The bioengineered organ germ is the 3D cell manipulation method approach that is
mimicking the organogenesis process by inducing the reciprocal mesenchymal–epithelial
cells interaction in a similar way that occurs in a natural tooth development. Taking into ac-
count the type of cell sources, embryonic and non-embryonic tooth germ-derived epithelial
cells in combination with mesenchymal stem cells were used for whole tooth regeneration.
The mouse embryonic tooth germ cells represent suitable candidates for the regenera-
tion of the whole tooth since they allow the growth of the functional tooth in a relatively
short time via the reciprocal mesenchymal–epithelial cells interaction in a type I collagen
gel [199]. The primary enamel knot acts as a second signaling center and represents an
important step in the reconstitution of functional germ cells. Following the dissociation
of the enamel organ and the dental mesenchyme cells from the first lower molars of the
mouse in the early stage of the cap (embryonic day 14-E14), the resulting cells were cultured
and reassociated into in vitro intact dental mesenchyme or dissociated mesenchymal cells.
Although both types of experiments yielded the tooth development, the intact mesenchy-
mal tissue led to a faster development, most likely due to its ability to memorize the cell
history. Nevertheless, the equal duration of progression in the first stages of epithelial
histogenesis for both experiments highlights that the history of early-stage re-associations
is not memorized by mesenchymal tissue [215].
Ikeda et al. formulated the main concepts as regards tooth regeneration, i.e., in vitro
manipulation of single cells, in vivo transplantation, identification of the cell sources, and
determining the tooth morphology concepts to be realized by means of several technologies.
These technologies included scaffold tissue engineering combined with cell aggregation,
transplantation of the tooth germ or bioengineered tooth in a mature oral environment,
identification of the inductive tooth cells belonging to dental and adult tissues, and super-
vising the signaling cascades that control tooth morphology in conjunction with scaffold
tissue engineering [216]. Furthermore, Ishida et al. [217] reported the influence of the
contact area between mesenchymal cells and epithelial cells, cell proliferation, and sonic
hedgehog (Shh) expression of the inner enamel epithelium on the cusps number and crown
width of a bioengineered molar tooth.
The transfer of a bioengineered tooth germ, rebuilt from an E14.5 embryonic day
tooth germ derived from mesenchymal and epithelial cells, into the alveolar bone of an
adult mouse yielded the obtaining of a fully functional tooth with appropriate structure,
adequate hardness of mineralized tissues to withstand chewing activity, as well as ability
to react to experimental orthodontic treatment and harmful stimulation in association
with the tissues from the oral and maxillofacial regions [218]. One research article [219]
Materials 2021, 14, 2558 24 of 35

evidenced the ability of embryonic dental mesenchyme tissues of human origin, attained
from the cap stage, to determine tooth formation only in the presence of the humans or
mice non-dental epithelium cells. Furthermore, the dental mesenchyme cells from the bell
stage succeeded to transform both human keratinocyte stem cells and mouse embryonic
secondary arch epithelium into enamel-secreting ameloblasts.
Non-embryonic cells have been also used as cell sources in the formation of tooth
through bioengineered organ germ method [201]. Although additional research is needed
to establish more consistent protocols, the adult stem bone marrow mesenchymal (BM-
MSCs) cells are of particularly interest for the regeneration of the whole tooth since may be
used as substitutes of dental mesenchymal cells [208,220]. BM-MSCs can be differentiated
into various types of cells, such as ameloblast-like ones, can increase the level of the
odontogenic gene’s expression and can be used for the whole tooth regeneration after
recombining with the embryonic oral epithelium cells [221]. The dental epithelium rebuilt
with BM-MSCs succeeded to produce the odontogenesis inductive signals at around E10,
thus triggering the formation of a tooth de nuovo [222]. It was established that BM-MSCs
yielded the formation of all types of mesenchymal derived cells from the tooth. The in vitro
culture led to the induction of early dental marker genes, while the in vivo one directed
the induction of dentin sialophosphoprotein (DSPP) within the BM-MSC aggregate cells
and, thus, influenced the formation of a tooth tissue. The implantation of rat BM-MSCs for
2 weeks into a pulpotomized pulp chamber containing a porous polyL-lactic acid scaffold
yielded the appearance of -galactosidase-expressing cells in the newly formed dentin
structures. In this way, it was revealed the differentiation potential of BM-MSCs in the
formation of new mineralized tissues [223].
Induced pluripotent stem cells (iPSCs) represent another type of cell source for bioengi-
neered tooth due to their characteristics analogous with the embryonic stem cells (ESCs),
i.e., self-renewal capability, differentiation into germ layers, large-scale expansion [224].
Furthermore, the use of iPSCs is beneficial since the problems related with the immunolog-
ical rejection and/or ethical controversy can be avoided. Wen et al. reported the capability
of iPSCs to differentiate toward odontogenic cells by using a recombinant tooth germ
model containing mouse iPSC, epithelial and mesenchymal cells, transplanted for 4 weeks
into a mouse subrenal capsule [224]. Otsu et al. [225] evidenced the ability of the neural
crest-like cells (NCLC) derived from mouse iPSCs to differentiate within odontogenic
mesenchymal cells and odontoblasts after the stimulation with dental epithelium. The
potential of the epithelial sheets obtained from human urine induced pluripotent stem cells
(ifhU-iPSCs) to substitute the tooth germ in the presence of mouse dental mesenchyme and
to generate in vivo tooth-like structures was reported by Cai et al. [140]. The iPSCs-derived
ameloblasts were found to possess the capability to secrete enamel, although of a lessened
hardness, with elastic properties comparable to regular human tooth.
Odontoblast- and ameloblast-like cells were efficaciously generated by culturing the
mouse iPSCs into an ameloblasts serum-free conditioned medium containing the BMP4
bone morphogenetic protein [226]. During tooth morphogenesis, the iPSCs differentiation
induced by the presence of BM4 was regulated by specific key proteins and genes.

6.3. Whole Tooth Regeneration through Tissue Engineering Approach

The basic principle regarding the tissue engineering approach of the whole tooth
relies on developing biological alternatives able to be involved in the tooth formation
that contain cells, scaffolds, and bioactive agents in order to obtain organs and tissues
analogous to native human ones. Replacement of the entire tissue of an affected organ
can be done using 3D structures cultured in vitro with cells. It is assumed that future
technologies will reconstitute in vitro the entire organ to substitute the dysfunctional tissue.
Regeneration of the whole tooth requires accompanying the cells with adequate scaffolds,
usually based on synthetic or natural polymers. The ideal scaffold should be biocompatible,
conductive, and provide adequate physical properties and chemical stability while assuring
Materials 2021, 14, 2558 25 of 35

cell compatibility and proliferation, adhesion performance, mechanical strength, controlled

degradation, and specific nano- and micro-scaled topology [111].
As regards the scaffold materials used for the tissue engineering process of the whole
tooth, one can mention hydrogels based on gelatin methacrylate [227–229], gelatin methacry-
lamide [230] and methacryloyl [231], polyglycolate [232], and poly (D,Llactide-co-glycolide)
in combination with gelatin [233], silk [234], and decellularized scaffolds [235,236]. In these
research studies, the cultured dissociated porcine and dental mesenchymal cells were
combined, seeded on different biodegradable scaffolds, i.e., polyglycolate and poly-L-
lactate-coglycolate or polyglycolate–poly-L-lactate transplanted and grown in rat hosts.
Instead of the formation of one tooth with shape and size analogous to the scaffold, the
appearance of small tooth crowns containing dentin, odontoblasts, Hertwig’s epithelial root
sheath, a pre-defined pulp chamber, and enamel organ was noticed throughout the whole
implant. In a similar manner, multiple teeth with both normal and irregular morphologies,
as well as tooth root-like structures were generated after seeding the dental epithelial and
mesenchymal pig tooth bud cells onto collagen sponge scaffolds [237].
The encapsulation of postnatal dental cells within gelatin methacrylate hydrogel
scaffolds yielded the obtaining of a new type of biomimetic tooth buds [227]. These
structures facilitated the dental epithelial–dental epithelial, dental mesenchymal–dental
mesenchymal, and dental epithelial–dental mesenchymal cell interactions, differentia-
tion of ameloblasts and odontoblasts and construction of a bioengineered tooth with
predictable shape and size. A 3D biomimetic tooth bud comprising successive layers of
gelatin methacrylamide hydrogels seeded with dental mesenchymal and dental epithelial
cells favorized the differentiation of the dental cells into odontoblasts and ameloblasts,
thus triggering the development of a bioengineered tooth of expected size and shape [230].
Smith et al. [231] reported the obtaining of vastly cellularized dental buds which
exhibited distinctive features common to those of natural ones, such as enamel knot signal-
ing centers, niche of dental epithelial stem cells, mineralized dental tissues, and transient
amplification cells. The tooth development was demonstrated by using postnatal dental
cells encapsulated inside gelatin methacryloyl hydrogel scaffolds and further implanted
beneath the skin of some immunocompromised rats. Isolated and dissociated E14 mice
tooth germs seeded onto a polyglycolate 3D scaffold and further implanted inside a mice
kidney capsule were able to produce a molar tooth. Starting with the fifth day, the dissoci-
ated cells were able to form an initial tooth germ, process followed by the development of
dentin covered with enamel.
A 5% gelatin methacrylate hydrogel supported the creation of extremely vascularized
and cellularized pulp-like tissues containing human dental pulp and human umbilical
vein endothelial cells in vivo implanted in tooth root segments, attachment of the cells
to the inner surface of the dentin from the tooth root, and formation of reparative dentin
matrix [228].
By simulating a complete extracellular matrix microenvironment for stem cells, gelatin
electrospun sheets combined with treated dentin matrix substrates succeeded to promote
the regeneration of the tooth root [233]. As follows, two composites, i.e., one containing
aligned poly (D,Llactide-co-glycolide)/gelatin electrospun sheets and treated dentin matrix,
and another one comprising native dental pulp extracellular matrix and treated dentin
matrix were fabricated for the regeneration of periodontium and dental pulp. Such types
of scaffolds were able to simulate the properties of an extracellular matrix, thus facilitating
the differentiation of the dental stem cells and formation of odontoblast-like layers between
the dental pulp-like tissues and pre-dentin matrix, blood vessels, cellular cementum and
periodontal ligament-like tissues.
Taking into account the difficulties encountered when designing synthetic scaffolds
with desired properties to mimic ECM properties and to enhance the tooth regeneration,
decellularized scaffolds were used as alternatives. Several approaches, such as chemical,
physical and enzymatic methods in which cell membranes were interrupted and cleansed
were used to obtain decellularized scaffolds. Different methods for obtaining decellularized
Materials 2021, 14, 2558 26 of 35

dental tissues from porcine molar tooth buds, while preserving the extracellular matrix
proteins such as fibronectin, collagen, and fibronectin in tooth structures formed in early
stages were reported by Traphagen et al. [235]. The decellularized tooth seeded with cells
presented a higher amount of collagen as compared to the unseeded references.
Zhang et al. [236] employed a decellularized tooth bud scaffold in order to induce
the whole tissue regeneration. The decellularized scaffold was seeded with human dental
pulp cells, porcine dental epithelial cells, as well as human umbilical vein endothelial
cells. The scaffold displayed a higher degree of the cellular activity and induced the cell
differentiation that allowed the regeneration of the whole tooth.
Four types of hexafluoroisopropanol-based silk scaffolds having two different pore
diameter dimensions, with or without peptides (arginine-glycine-aspartic acid) were used
to fabricate vigorous tooth bud-like tissues derived from mineralized osteodentin cells [234].
The silk scaffolds with larger pores and peptides yielded a better generation of mineralized
dental tissues as compared to the ones with smaller pores and which did not contain peptides.
Song et al. [238] reported the obtaining of scaffolds based on decellularized human
dental pulp as supports for differentiation and proliferation of the stem cells from apical
papilla. As follows, subsequent recellularization of the scaffold yielded proliferation and
differentiation of SCAPs into odontoblast-like cells nearby dentinal walls and regeneration
of the whole tooth.

7. Conclusions and Perspectives

The present review presented the recent advances in the stem cell-based regeneration
strategies for hard dental tissues. A special emphasis was given to the regeneration of
the whole tooth, being detailed the main approaches in designing suitable scaffolds that
provide an appropriate environment for the development of stem cells. Regardless of the
chosen method, i.e., scaffold-based or scaffold-free approach, stem cells were able to fully
contribute to the regeneration of the hard dental tissues and of the whole tooth. Despite the
development of the scaffold-free tissue engineering technique as a powerful approach for
tooth regeneration, the use of suitable biomaterial-based scaffolds still represents the main
approach to regenerate dentin and cementum hard dental tissues, as well as the whole tooth.
So far, the efficiency of stem cells-based regeneration of enamel was partially limited by the
high level of cells interconnectivity and specialization required for enamel regeneration.
Although significant progress has been made in the regeneration of hard dental tissues,
there are still some major obstacles as regards the application of the “processed tooth”
concept in clinical practice. To achieve this ambitious task, the architectural design of the
bioengineered teeth will have to take into considerations several important aspects, i.e., (i)
identification of the master genes from the gene regulatory networks responsible for the
induction and tooth regeneration, (ii) controlling the bioengineered teeth parameters (shape,
size and color) in order to correctly and accurately restore the missing tooth, even under the
acid attack from the oral cavity, (iv) developing of new types of cell culture techniques able
to achieve fully functionalized regenerated tooth comprising vascularization, innervation
and supporting tissues, (iv) applying innovative approaches able to induce in vivo enamel
regeneration, and (v) maintaining of the long term oral health and preventing the failure of
the regenerated tooth.

Author Contributions: L.S.: Conceptualization, Validation, Methodology, Writing—Original Draft.

G.C.: Software, Resources: M.O.: Investigation, Resources, Writing—Original Draft. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Materials 2021, 14, 2558 27 of 35

Acknowledgments: Not applicable.

Conflicts of Interest: The authors declare no conflict of interest.

References
1. Beniash, E.; Stifler, C.A.; Sun, C.-Y.; Jung, G.S.; Qin, Z.; Buehler, M.J.; Pupa, U.P.; Gilbert, A. The hidden structure of human
enamel. Nat. Commun. 2019, 10, 4383. [CrossRef] [PubMed]
2. Murata, M.; Akazawa, T.; Mitsugi, M.; Arafat, M.; Um, I.W.; Minamida, Y.; Kim, K.W.; Kim, Y.K.; Sun, Y.; Qin, C. Autograft of
dentin materials for bone regeneration. Adv. Biomater. Sci. Biomed. Appl. 2013, 15, 391–403.
3. Yamamoto, T.; Hasegawa, T.; Yamamoto, T.; Hongo, H.; Amizuka, N. Histology of human cementum: Its structure, function, and
development. Jpn. Dent. Sci. Rev. 2016, 52, 63–74. [CrossRef] [PubMed]
4. Nazir, M.A. Prevalence of periodontal disease, its association with systemic diseases and prevention. Int. J. Health Sci. 2017,
11, 72–80.
5. Lacruz, R.S.; Habelitz, S.; Wright, J.T.; Paine, M.L. Dental enamel formation and implications for oral health and disease. Physiol.
Rev. 2017, 97, 939–993. [CrossRef] [PubMed]
6. Conrads, G.; About, I. Pathophysiology of dental caries. In Caries Excavation, Evolution of Treating Cavitated Carious Lesions; Karger
Medical and Scientific Publishers: Berlin, Germany, 2018; pp. 1–10.
7. Howard, D.; Buttery, L.D.; Shakesheff, K.M.; Roberts, S.J. Tissue engineering, strategies, stem cells and scaffolds. J. Anat. 2008,
213, 66–72. [CrossRef]
8. Yen, A.H.; Sharpe, P.T. Stem cells and tooth tissue engineering. Cell Tissue Res. 2008, 331, 359–372. [CrossRef] [PubMed]
9. Sharma, S.; Srivastava, D.; Grover, S.; Sharma, V. Biomaterials in tooth tissue engineering, a review. J. Clin. Diagn. Res. JCDR 2014,
8, 309–315. [CrossRef]
10. Otsu, K.; Kumakami-Sakano, M.; Fujiwara, N.; Kikuchi, K.; Keller, L.; Lesot, H.; Hidemitsu, H. Stem cell sources for tooth
regeneration: Current status and future prospects. Front. Physiol. 2014, 5, 36. [CrossRef]
11. Rider, P.; Kačarević, Ž.P.; Alkildani, S.; Retnasingh, S.; Barbeck, M. Bioprinting of tissue engineering scaffolds. J. Tissue Eng. 2018,
9, 1–16. [CrossRef]
12. Baranova, J.; Büchner, D.; Götz, W.; Schulze, M.; Tobiasch, E. Tooth formation: Are the hardest tissues of human body hard to
regenerate? Int. J. Mol. Sci. 2020, 21, 4031. [CrossRef] [PubMed]
13. Ahmed, G.M.; Abouauf, E.A.; AbuBakr, N.; Dörfer, C.E.; El-Sayed, K.F. Tissue engineering approaches for enamel, dentin, and
pulp regeneration: An update. Stem Cells Int. 2020, 2020, 1–15. [CrossRef] [PubMed]
14. Kim, M.G.; Park, C.H. Tooth-supporting hard tissue regeneration using biopolymeric material fabrication strategies. Molecules
2020, 25, 4802. [CrossRef] [PubMed]
15. Liu, G.; David, B.T.; Trawczynski, M.; Fessler, R.G. Advances in pluripotent stem cells: History, mechanisms, technologies, and
applications. Stem Cell Rev. 2019, 16, 3–32. [CrossRef] [PubMed]
16. Xu, M.; He, J.; Zhang, C.; Xu, J.; Wang, Y. Strategies for derivation of endothelial lineages from human stem cells. Stem Cell Res.
Ther. 2019, 10. [CrossRef] [PubMed]
17. Orbay, H.; Tobita, M.; Mizuno, H. Mesenchymal stem cells isolated from adipose and oTher. tissues: Basic biological properties
and clinical applications. Stem Cells Int. 2012, 2012, 1–9. [CrossRef] [PubMed]
18. Gan, L.; Liu, Y.; Cui, D.; Pan, Y.; Zheng, L.; Wan, M. Dental tissue-derived human mesenchymal stem cells and their potential in
therapeutic application. Stem Cells Int. 2020, 2020, 1–17. [CrossRef]
19. Bianco, P.; Robey, P.G.; Simmons, P.J. Mesenchymal stem cells: Revisiting history, concepts, and assays. Cell Stem Cell 2008,
2, 313–319. [CrossRef] [PubMed]
20. Zhai, Q.; Dong, Z.; Wang, W.; Li, B.; Jin, Y. Dental stem cell and dental tissue regeneration. Front. Med. 2018, 13, 152–159.
[CrossRef]
21. Zhang, Y.; Chen, Y. Bioengineering of a human whole tooth: Progress and challenge. Cell Regen. 2014, 3, 1–3. [CrossRef]
22. Sahab-Negah, S.; Hajali, V.; Moradi, H.R.; Gorji, A. The impact of estradiol on neurogenesis and cognitive functions in Alzheimer’s
disease. Cell. Mol. Neurobiol. 2020, 40, 283–299. [CrossRef] [PubMed]
23. Mitsiadis, T.A.; Feki, A.; Papaccio, G.; Catón, J. Dental pulp stem cells, niches, and notch signaling in tooth injury. Adv. Dent. Res.
2011, 23, 275–279. [CrossRef] [PubMed]
24. Chan, B.P.; Leong, K.W. Scaffolding in tissue engineering: General approaches and tissue-specific considerations. Eur. Spine J.
2008, 17, 467–479. [CrossRef] [PubMed]
25. Zakrzewski, W.; Dobrzyński, M.; Szymonowicz, M.; Rybak, Z. Stem cells: Past, present, and future. Stem Cell Res. Ther. 2019, 10.
[CrossRef] [PubMed]
26. Huang, G.T.-J.; Gronthos, S.; Shi, S. Mesenchymal stem cells derived from dental tissues vs. those from oTher. sources: Their
biology and role in regenerative medicine. J. Dent. Res. 2009, 88, 792–806. [CrossRef] [PubMed]
27. Kyurkchiev, D. Secretion of immunoregulatory cytokines by mesenchymal stem cells. World J. Stem Cells 2014, 6, 552. [CrossRef]
[PubMed]
28. Yang, X.; Li, L.; Xiao, L.; Zhang, D. Recycle the dental fairy’s package: Overview of dental pulp stem cells. Stem Cell Res. 2018, 9.
[CrossRef] [PubMed]
Materials 2021, 14, 2558 28 of 35

29. Kawashima, N.; Okiji, T. Odontoblasts: Specialized hard-tissue-forming cells in the dentin-pulp complex. Congenit. Anom. 2016,
56, 144–153. [CrossRef]
30. Ikeda, E.; Nakagawa, M.; Ogawa, M.; Takeo, M.; Tsuji, T. Functional tooth regeneration as a next-generation therapy. J. Dent. Oral
Disord. 2020, 6, 114.
31. Nanci, A. Ten Cate’s Oral Histology: Development, Structure, and Function, 8th ed.; Mosby: Maryland Heights, MO, USA, 2003; p. 400.
32. Stevens, M.M. Exploring and engineering the cell surface interface. Science 2005, 310, 1135–1138. [CrossRef]
33. Chen, F.-M.; An, Y.; Zhang, R.; Zhang, M. New insights into and novel applications of release technology for periodontal
reconstructive therapies. J. Control. Release 2011, 149, 92–110. [CrossRef] [PubMed]
34. Griffin, M.F.; Butler, P.E.; Seifalian, A.M.; Kalaskar, D.M. Control of stem cell fate by engineering their micro and nanoenvironment.
World J. Stem Cells 2015, 26, 37–50. [CrossRef] [PubMed]
35. Aurrekoetxea, M.; Irastorza, I.; García-Gallastegui, P.; Jiménez-Rojo, L.; Nakamura, T.; Yamada, Y.; Ibarretxe, G.; Unda, F.J.
Wnt/ -catenin regulates the activity of epiprofin/Sp6, SHH, FGF, and BMP to coordinate the stages of odontogenesis. Front. Cell
Dev. Biol. 2016, 4, 25. [CrossRef] [PubMed]
36. Seppala, M.; Fraser, G.; Birjandi, A.; Xavier, G.; Cobourne, M. Sonic hedgehog signaling and development of the dentition. J. Dev.
Biol. 2017, 5, 6. [CrossRef] [PubMed]
37. Saygin, N.E.; Tokiyasu, Y.; Giannobile, W.V.; Somerman, M.J. Growth factors regulate expression of mineral associated genes in
cementoblasts. J. Periodontol. 2000, 71, 1591–1600. [CrossRef] [PubMed]
38. Ngo, V.A.; Jung, J.-Y.; Koh, J.-T.; Oh, W.-M.; Hwang, Y.-C.; Lee, B.-N. Leptin induces odontogenic differentiation and angiogenesis
in human dental pulp cells via activation of the mitogen-activated protein kinase signaling pathway. J. Endod. 2018, 44, 585–591.
[CrossRef] [PubMed]
39. Luder, H.U.; Graf, D. Bone morphogenetic protein 2 coordinates early tooth mineralization. J. Dent. Res. 2018, 97, 835–843.
40. Ishikawa, Y.; Nakatomi, M.; Ida-Yonemochi, H.; Ohshima, H. Quiescent adult stem cells in murine teeth are regulated by Shh
signaling. Cell Tissue Res. 2017, 369, 497–512. [CrossRef]
41. Tonk, C.; Witzler, M.; Schulze, M.; Tobiasch, E. Mesenchymal stem cells. In Essential Current Concepts in Stem Cell Biology;
Brand-Saberi, B., Ed.; Springer: Berlin, Germany, 2020; pp. 21–39.
42. Witzler, M.; Alzagameem, A.; Bergs, M.; Khaldi-Hansen, B.E.; Klein, S.E.; Hielscher, D.; Kamm, B.; Kreyenschmidt, J.; Tobiasch, E.;
Schulze, M. Lignin-derived biomaterials for drug release and tissue engineering. Molecules 2018, 23, 1885. [CrossRef]
43. Khaldi-Hansen, E.B.; El-Sayed, F.; Tobiasch, E.; Witzleben, S.; Schulze, M. Functionalized 3D scaffolds for template- mediated
biomineralization in bone regeneration. Front. Stem Cell Regen. Med. Res. 2017, 4, 3–58.
44. Leiendecker, A.; Witzleben, S.; Schulze, M.; Tobiasch, E. Template-mediated biomineralization for bone tissue engineering. Curr.
Stem Cell Res. T 2016, 12, 103–123. [CrossRef] [PubMed]
45. Bains, P.S.; Sidhu, S.S.; Bahraminasab, M.; Prakash, C. Materials Horizons: From Nature to Nanomaterials; Springer Nature Pte Ltd.:
Singapore, 2019.
46. Horst, O.V.; Chavez, M.G.; Jheon, A.H.; Desai, T.; Klein, O.D. Stem cell and biomaterials reseArch. in dental tissue engineering
and regeneration. Dent. Clin. N. Am. 2012, 56, 495–520. [CrossRef] [PubMed]
47. Yang, Y.; Ritchie, A.C.; Everitt, N.M. Comparison of glutaraldehyde and procyanidin cross-linked scaffolds for soft tissue
engineering. Mat. Sci. Eng. C 2017, 80, 263–273. [CrossRef] [PubMed]
48. Coyac, B.R.; Chicatun, F.; Hoac, B.; Nelea, V.; Chaussain, C.; Nazhat, S.N.; McKee, M.D. Mineralization of dense collagen hydrogel
scaffolds by human pulp cells. J. Dent. Res. 2013, 92, 648–654. [CrossRef] [PubMed]
49. Abou Neel, E.A.; Cheema, U.; Knowles, J.C.; Brown, R.A.; Nazhat, S.N. Use of multiple unconfined compression for control of
collagen gel scaffold density and mechanical properties. Soft Matter 2006, 2, 986. [CrossRef] [PubMed]
50. Ashammakhi, N.; Ahadian, S.; Xu, C.; Montazerian, H.; Ko, H.; Nasiri, R.; Barros, N.; Khademhosseinie, A. Bioinks and
bioprinting technologies to make heterogeneous and biomimetic tissue constructs. Mater. Today Bio. 2019, 1, 100008. [CrossRef]
[PubMed]
51. Silver, F.; Pins, G. Cell growth on collagen: A review of tissue engineering using scaffolds containing extracellular matrix. J. Long
Term Eff. Med. Implant. 1992, 2, 67–80.
52. Zhu, L.; Luo, D.; Liu, Y. Effect of the nano/microscale structure of biomaterial scaffolds on bone regeneration. Int. J. Oral Sci.
2020, 12. [CrossRef]
53. Rozario, T.; DeSimone, D.W. The extracellular matrix in development and morphogenesis: A dynamic view. Dev. Biol. 2010,
341, 126–140. [CrossRef]
54. Wang, Y.-F.; Wang, C.-Y.; Wan, P.; Wang, S.-G.; Wang, X.-M. Comparison of bone regeneration in alveolar bone of dogs on
mineralized collagen grafts with two composition ratios of nano-hydroxyapatite and collagen. Regen. Biomater. 2015, 3, 33–40.
[CrossRef]
55. Sancilio, S.; Gallorini, M.; Di Nisio, C.; Marsich, E.; Di Pietro, R.; Schweikl, H. Alginate/hydroxyapatite-based nanocomposite
scaffolds for bone tissue engineering improve dental pulp biomineralization and differentiation. Stem Cells Int. 2018, 2018, 1–13.
[CrossRef] [PubMed]
56. Tohamy, K.M.; Mabrouk, M.; Soliman, I.E.; Beherei, H.H.; Aboelnasr, M.A. Novel alginate/hydroxyethyl cellulose/hydroxyapatite
composite scaffold for bone regeneration: In vitro cell viability and proliferation of human mesenchymal stem cells. Int. J. Biol.
MacroMol. 2018, 112, 448–460. [CrossRef] [PubMed]
Materials 2021, 14, 2558 29 of 35

57. Turco, G.; Marsich, E.; Bellomo, F.; Semeraro, S.; Donati, I.; Brun, F.; Grandolfo, M.; Accardo, A.; Paolleti, S. Al-
ginate/hydroxyapatite biocomposite for bone ingrowth: A trabecular structure with high and isotropic connectivity.
Biomacromolecules 2009, 10, 1575–1583. [CrossRef] [PubMed]
58. Farzin, A.; Bahrami, N.; Mohamadnia, A.; Mousavi, S.; Gholami, M.; Ai, J.; Moayeri, R.S. Scaffolds in dental tissue engineering: A
review. Arch. NeuroSci. 2019, 7, e97014. [CrossRef]
59. Masuda, K.; Sah, R.L.; Hejna, M.J.; Thonar, E.J. A novel two-step method for the formation of tissue-engineered cartilage by
mature bovine chondrocytes: The alginate-recovered-chondrocyte (ARC) method. J. Orthop. Res. 2003, 21, 139–148. [CrossRef]
60. Ahmed, T.A.E.; Dare, E.V.; Hincke, M. Fibrin: A versatile scaffold for tissue engineering applications. Tissue Eng. Part B Rev. 2008,
14, 199–215. [CrossRef] [PubMed]
61. Al Kayal, T.; Losi, P.; Pierozzi, S.; Soldani, G. A new method for fibrin-based electrospun/sprayed scaffold fabrication. Sci. Rep.
2020, 10. [CrossRef] [PubMed]
62. Litvinov, R.I.; Weisel, J.W. Fibrin mechanical properties and their structural origins. Matrix Biol. 2017, 60–61, 110–123. [CrossRef]
63. Kattula, S.; Byrnes, J.R.; Wolberg, A.S. Fibrinogen and fibrin in hemostasis and thrombosis. Arterioscler. Thromb. Vasc. Biol. 2017,
37, e13–e21. [CrossRef]
64. Wang, Z.S.; Feng, Z.H.; Wu, G.F.; Bai, S.-Z.; Dong, Y.; Chen, F.-M.; Zhao, Y.-M. The use of platelet-rich fibrin combined with
periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering. Sci. Rep. 2016, 6, 28126.
[CrossRef] [PubMed]
65. Sybil, D.; Sawai, M.; Faisal, M.; Singh, S.; Jain, V. Platelet-rich fibrin for hard- and soft-tissue healing in mandibular third molar
extraction socket. Ann. Maxillofac. Surg. 2020, 10, 102–107. [PubMed]
66. Bujoli, B.; Scimeca, J.C.; Verron, E. Fibrin as a multipurpose physiological platform for bone tissue engineering and targeted
delivery of bioactive compounds. Pharmaceutics 2019, 11, 556. [CrossRef] [PubMed]
67. Zhao, H.; Ma, L.; Gao, C.; Wang, J.; Shen, J. Fabrication and properties of injectable -tricalcium phosphate particles/fibrin gel
composite scaffolds for bone tissue engineering. Mater. Sci. Eng. C 2009, 29, 836–842. [CrossRef]
68. Liu, M.; Zeng, X.; Ma, C.; Yi, H.; Ali, Z.; Mou, X.; Li, S.; Deng, Y.; He, N. Injectable hydrogels for cartilage and bone tissue
engineering. Bone Res. 2017, 5, 17014. [CrossRef] [PubMed]
69. Zhou, H.; Chen, W.; Weir, M.D.; Xu, H.H.K. Biofunctionalized calcium phosphate cement to enhance the attachment and
osteodifferentiation of stem cells released from fast-degradable alginate-fibrin microbeads. Tissue Eng. Part A 2012, 18, 1583–1595.
[CrossRef] [PubMed]
70. Chen, W.; Zhou, H.; Weir, M.D.; Bao, C.; Xu, H.H.K. Umbilical cord stem cells released from alginate–fibrin microbeads
inside macroporous and biofunctionalized calcium phosphate cement for bone regeneration. Acta Biomater. 2012, 8, 2297–2306.
[CrossRef]
71. Lv, J.; Xiu, P.; Tan, J.; Jia, Z.; Cai, H.; Liu, Z. Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled
release of BMP-2 and VEGF: Implantation of electron beam melting-fabricated porous Ti6 Al4 V scaffolds incorporating growth
factor-doped fibrin glue. BioMed. Mater. 2015, 10, 035013. [CrossRef]
72. Kopf, B.S.; Schipanski, A.; Rottmar, M.; Berner, S.; Maniura-Weber, K. Enhanced differentiation of human osteoblasts on Ti
surfaces pre-treated with human whole blood. Acta Biomater. 2015, 19, 180–190. [CrossRef]
73. Nichol, J.W.; Koshy, S.T.; Bae, H.; Hwang, C.M.; Yamanlar, S.; Khademhosseini, A. Cell-laden microengineered gelatin methacry-
late hydrogels. Biomaterials 2010, 31, 5536–5544. [CrossRef]
74. Zhao, Y.; Chen, X.; Bai, S.; Li, B.; Liu, H.; Wu, G.; Liu, S.; Zhao, Y. Fabrication of gelatin methacrylate/nanohydroxyapatite
microgel arrays for periodontal tissue regeneration. Int. J. Nanomed. 2016, 11, 4707–4718. [CrossRef]
75. Monteiro, N.; Thrivikraman, G.; Athirasala, A.; Tahayeri, A.; França, C.M.; Ferracane, J.L.; Bertassoni, L.E. Photopolymerization
of cell-laden gelatin methacryloyl hydrogels using a dental curing light for regenerative dentistry. Dent. Mater. 2018, 34, 389–399.
[CrossRef] [PubMed]
76. Cacciotti, I.; Chronopoulou, L.; Palocci, C.; Amalfitano, A.; Cantiani, M.; Cordaro, M.; Lajolo, C.; Calla, C.; Boninsegna, A.;
Lucchetti, D.; et al. Controlled release of 18- -glycyrrhetic acid by nanodelivery systems increases cytotoxicity on oral carcinoma
cell line. Nanotechnology 2018, 29, 285101. [CrossRef] [PubMed]
77. Kumar, S.K.; Krishnamoorti, R. Nanocomposites: Structure, phase behavior, and properties. Annu. Rev. Chem. BioMol. Eng. 2010,
1, 37–58. [CrossRef] [PubMed]
78. Keles, H.; Naylor, A.; Clegg, F.; Sammon, C. Investigation of factors influencing the hydrolytic degradation of single PLGA
microparticles. Polym. Degrad. Stab. 2015, 119, 228–241. [CrossRef]
79. Sun, X.; Xu, C.; Wu, G.; Ye, Q.; Wang, C. Poly(lactic-co-glycolic acid): Applications and future prospects for periodontal tissue
eegeneration. Polymers 2017, 9, 189. [CrossRef] [PubMed]
80. Pan, Z.; Ding, J. Poly(lactide-co-glycolide) porous scaffolds for tissue engineering and regenerative medicine. Interface Focus 2012,
2, 366–377. [CrossRef] [PubMed]
81. Song, R.; Murphy, M.; Li, C.; Ting, K.; Soo, C.; Zheng, Z. Current development of biodegradable polymeric materials for
biomedical applications. Drug Des. Devel. Ther. 2018, 12, 3117–3145. [CrossRef] [PubMed]
82. Ulery, B.D.; Nair, L.S.; Laurencin, C.T. Biomedical applications of biodegradable polymers. J. Polym. Sci. Part B Polym. Phys. 2011,
49, 832–864. [CrossRef]
Materials 2021, 14, 2558 30 of 35

83. Arnold, M.M.; Gorman, E.M.; Schieber, L.J.; Munson, E.J.; Berkland, C. Nano Cipro encapsulation in monodisperse large porous
PLGA microparticles. J. Control. Release 2007, 121, 100–109. [CrossRef]
84. Narayan, D.; Venkatraman, S.S. Effect of pore size and interpore distance on endothelial cell growth on polymers. J. BioMed.
Mater. Res. Part A 2008, 87, 710–718. [CrossRef] [PubMed]
85. Spalazzi, J.P.; Vyner, M.C.; Jacobs, M.T.; Moffat, K.L.; Lu, H.H. Mechanoactive scaffold induces tendon remodeling and expression
of fibrocartilage markers. Clin. Orthop. Relat. Res. 2008, 466, 1938–1948. [CrossRef] [PubMed]
86. Jabbarzadeh, E.; Starnes, T.; Khan, Y.M.; Jiang, T.; Wirtel, A.J.; Deng, M.; Lv, Q.; Nair, L.S.; Doty, S.B.; Laurencin, C.T. Induction of
angiogenesis in tissue-engineered scaffolds designed for bone repair: A combined gene therapy-cell transplantation approach.
Proc. Natl. Acad. Sci. USA 2008, 105, 11099–11104. [CrossRef] [PubMed]
87. Chung, H.J.; Kim, I.K.; Kim, T.G.; Park, T.G. Highly Open Porous Biodegradable Microcarriers: In Vitro Cultivation of Chondro-
cytes for Injectable Delivery. Tissue Eng. Part A 2008, 14, 607–615. [CrossRef] [PubMed]
88. Zhu, X.H.; Lee, L.Y.; Jackson, J.S.H.; Tong, Y.W.; Wang, C.-H. Characterization of porous poly (D,L-lactic-co-glycolic acid) sponges
fabricated by supercritical CO2 gas-foaming method as a scaffold for three-dimensional growth of Hep3B cells. Biotechnol. BioEng.
2008, 100, 998–1009. [CrossRef]
89. Ge, Z.; Wang, L.; Heng, C.B.; Tian, X.-F.; Lu, K.; Fan, V.T.W.; Yeo, J.F.; Cao, T.; Tan, E. Proliferation and differentiation of human
osteoblasts within 3D printed poly-lactic-co-glycolic acid scaffolds. J. Biomater. Appl. 2009, 23, 533–547.
90. Bashur, C.A.; Dahlgren, L.A.; Goldstein, A.S. Effect of fiber diameter and orientation on fibroblast morphology and proliferation
on electrospun poly (d,l-lactic-co-glycolic acid) meshes. Biomaterials 2006, 27, 5681–5688. [CrossRef]
91. Kumbar, S.G.; Nukavarapu, S.P.; James, R.; Nair, L.S.; Laurencin, C.T. Electrospun poly(lactic acid-co-glycolic acid) scaffolds for
skin tissue engineering. Biomaterials 2008, 29, 4100–4107. [CrossRef]
92. Moffat, K.L.; Kwei, A.S.-P.; Spalazzi, J.P.; Doty, S.B.; Levine, W.N.; Lu, H.H. Novel nanofiber-based scaffold for rotator cuff repair
and augmentation. Tissue Eng. Part A 2009, 15, 115–126. [CrossRef]
93. Aviss, K.; Gough, J.; Downes, S. Aligned electrospun polymer fibRes. for skeletal muscle regeneration. Eur. Cells Mater. 2010,
19, 193–204. [CrossRef]
94. Yoon, J.J.; Chung, H.J.; Lee, H.J.; Park, T.G. Heparin-immobilized biodegradable scaffolds for local and sustained release of
angiogenic growth factor. J. BioMed. Mater. Res. Part A 2006, 79, 934–942. [CrossRef]
95. Perron, J.K.; Naguib, H.E.; Daka, J.; Chawla, A.; Wilkins, R. A study on the effect of degradation media on the physical and
mechanical properties of porous PLGA 85/15 scaffolds. J. BioMed. Mater. Res. Part B Appl. Biomater. 2009, 91, 876–886. [CrossRef]
[PubMed]
96. Leung, L.; Chan, C.; Baek, S.; Naguib, H. Comparison of morphology and mechanical properties of PLGA bioscaffolds. BioMed.
Mater. 2008, 3, 025006. [CrossRef] [PubMed]
97. Stoll, C.; John, T.; Endres, M.; Rosen, C.; Kaps, C.; Kohl, B.; Sittinger, M.; Ertel, W.; Schulze-Tanzil, G. Extracellular matrix
expression of human tenocytes in three-dimensional air-liquid and PLGA cultuRes. compared with tendon tissue: Implications
for tendon tissue engineering. J. Orthop. Res. 2010, 28, 1170–1177. [CrossRef] [PubMed]
98. Blackwood, K.A.; McKean, R.; Canton, I.; Freeman, C.O.; Franklin, K.L.; Cole, D.; Brook, J.; Farthing, P.; Rimmer, S.; Haycock, J.W.;
et al. Development of biodegradable electrospun scaffolds for dermal replacement. Biomaterials 2008, 29, 3091–3104. [CrossRef]
[PubMed]
99. Guarino, V.; Ambrosio, L. The synergic effect of polylactide fiber and calcium phosphate particle reinforcement in poly "-
caprolactone-based composite scaffolds. Acta Biomater. 2008, 4, 1778–1787. [CrossRef] [PubMed]
100. Kim, J.-J.; Bae, W.-J.; Kim, J.-M.; Kim, J.-J.; Lee, E.-J.; Kim, H.-W.; Kim, E.-C. Mineralized polycaprolactone nanofibrous matrix for
odontogenesis of human dental pulp cells. J. Biomater. Appl. 2013, 28, 1069–1078. [CrossRef]
101. Park, C.; Kim, K.-H.; Lee, Y.-M.; Seol, Y.-J. Advanced engineering strategies for periodontal complex regeneration. Materials 2016,
9, 57. [CrossRef] [PubMed]
102. Ahn, G.Y.; Ryu, T.-K.; Choi, Y.R.; Park, J.R.; Lee, M.J.; Choi, S.-W. Fabrication and optimization of nanodiamonds-composited
poly("-caprolactone) fibrous matrices for potential regeneration of hard tissues. Biomater. Res. 2018, 22, 16. [CrossRef]
103. Fuchs, A.; Youssef, A.; Seher, A.; Hartmann, S.; Brands, R.C.; Müller-Richter, U.D.A.; Kübler, A.C.; Linz, C. A new multilayered
membrane for tissue engineering of oral hard- and soft tissue by means of melt electrospinning writing and film casting—An
in vitro study. J. Craniomaxillofac. Surg. 2019, 47, 695–703. [CrossRef]
104. Xu, J.; Gou, L.; Zhang, P.; Li, H.; Qiu, S. Platelet-rich plasma and regenerative dentistry. Aust. Dent. J. 2020, 65, 131–142. [CrossRef]
105. Sequeira, D.B.; Oliveira, A.R.; Catarina, M.S.; Palma, P.J.; Ramos, C.; Figueiredo, M.H.; Santos, A.C.; Cardoso, A.L.; Peça, J.;
Santos, J.M. Regeneration of pulp-dentin complex using human stem cells of the apical papilla: In vivo. Clin. Oral Investig. 2021.
[CrossRef] [PubMed]
106. Cao, W.; Hench, L.L. Bioactive materials. Ceram. Int. 1996, 22, 493–507. [CrossRef]
107. Neo, M.; Kotani, S.; Nakamura, T.; Yamamuro, T.; Ohtsuki, C.; Kokubo, T.; Bando, Y. A comparative study of ultrastructuRes. of
the interfaces between four kinds of surface-active ceramic and bone. J. BioMed. Mater. Res. 1992, 26, 1419–1432. [CrossRef]
108. Cacciotti, I. Multisubstituted hydroxyapatite powders and coatings: The influence of the codoping on the hydroxyapatite
performances. Int. J. Appl. Ceram. Technol. 2019, 16, 1864–1884. [CrossRef]
109. Cacciotti, I. Bivalent cationic ions doped bioactive glasses: The influence of magnesium, zinc, strontium and copper on the
physical and biological properties. J. Mater. Sci. 2017, 52, 8812–8831. [CrossRef]
Materials 2021, 14, 2558 31 of 35

110. Nam, S.; Won, J.-E.; Kim, C.-H.; Kim, H.-W. Odontogenic differentiation of human dental pulp stem cells stimulated by the
calcium phosphate porous granules. J. Tissue Eng. 2011, 2011, 812547. [CrossRef]
111. Zhang, L.; Morsi, Y.; Wang, Y.; Li, Y.; Ramakrishna, S. Review scaffold design and stem cells for tooth regeneration. Jpn. Dent. Sci.
Rev. 2013, 49, 14–26. [CrossRef]
112. Fielding, G.A.; Bandyopadhyay, A.; Bose, S. Effects of silica and zinc oxide doping on mechanical and biological properties of 3D
printed tricalcium phosphate tissue engineering scaffolds. Dent. Mater. 2012, 28, 113–122. [CrossRef]
113. Tonomura, A.; Mizuno, D.; Hisada, A.; Kuno, N.; Ando, Y.; Sumit, Y.; Kagami, H. Differential effect of scaffold shape on dentin
regeneration. Ann. BioMed. Eng. 2010, 38, 1664–1671. [CrossRef]
114. Hench, L.L.; Jones, J.R.; Sepulveda, P. Bioactive materials for tissue engineering scaffolds. In Future Strategies for Tissue and Organ
Replacement; Imperial College Press: London, UK, 2002; pp. 3–24.
115. Fu, L.; Engqvist, H.; Xia, W. Glass–ceramics in dentistry: A review. Materials 2020, 13, 1049. [CrossRef]
116. El-Gendy, R.; Yang, X.B.; Newby, P.J.; Boccaccini, A.R.; Kirkham, J. Osteogenic differentiation of human dental pulp stromal cells
on 45S5 bioglass®based scaffolds in vitro and in vivo. Tissue Eng. Part A 2013, 19, 707–715. [CrossRef] [PubMed]
117. Pérez, R.A.; Won, J.-E.; Knowles, J.C.; Kim, H.-W. Naturally and synthetic smart composite biomaterials for tissue regeneration.
Adv. Drug Deliv. Rev. 2013, 65, 471–496. [CrossRef] [PubMed]
118. Liu, X.; Ma, P.X. Polymeric scaffolds for bone tissue engineering. Ann. BioMed. Eng. 2004, 32, 477–486. [CrossRef] [PubMed]
119. Ko, H.-F.; Sfeir, C.; Kumta, P.N. Novel synthesis strategies for natural polymer and composite biomaterials as potential scaffolds
for tissue engineering. Philos. Trans. A Math Phys. Eng. Sci. 2010, 368, 1981–1997. [CrossRef] [PubMed]
120. Henkel, J.; Woodru, M.A.; Epari, D.R.; Steck, R.; Glatt, V.; Dickinson, I.C.; Choong, P.F.; Schuetz, M.A.; Hutmacher, D.W. Bone
Regeneration Based on Tissue Engineering Conceptions—A 21st Century Perspective. Bone Res. 2013, 1, 216–248. [CrossRef]
121. Noori, A.; Ashrafi, S.J.; Vaez-Ghaemi, R.; Hatamian-Zaremi, A.; Webster, T.J. A review of fibrin and fibrin composites for bone
tissue engineering. Int. J. NanoMed. 2017, 12, 4937–4961. [CrossRef]
122. Zheng, L.; Yang, F.; Shen, H.; Hu, X.; Mochizuki, C.; Sato, M.; Wang, S.; Zhang, Y. The effect of composition of calcium phosphate
composite scaffolds on the formation of tooth tissue from human dental pulp stem cells. Biomaterials 2011, 32, 7053–7059.
[CrossRef]
123. Gandolfi, M.G.; Zamparini, F.; Degli Esposti, M.; Chiellini, F.; Aparicio, C.; Fava, F.; Fabbri, P.; Taddei, P.; Prati, C. Polylactic
acid-based porous scaffolds doped with calcium silicate and dicalcium phosphate dihydrate designed for biomedical application.
Mater. Sci. Eng. C 2018, 82, 163–181. [CrossRef]
124. Ho, C.C.; Fang, H.Y.; Wang, B.; Huang, T.H.; Shie, M.Y. The effects of biodentine/polycaprolactone three-dimensional-scaffold
with odontogenesis properties on human dental pulp cells. Int. Endod. J. 2018, 51, e291–e300. [CrossRef]
125. Liu, H.; Yan, X.; Pandya, M.; Luan, X.; Diekwisch, T.G.H. Daughters of the enamel organ: Development, fate, and function of the
stratum intermedium, stellate reticulum, and outer enamel epithelium. Stem Cells Dev. 2016, 25, 1580–1590. [CrossRef] [PubMed]
126. Pandya, M.; Lin, T.; Li, L.; Allen, M.J.; Jin, T.; Luan, X.; Diekwisch, T.G.H. Posttranslational amelogenin processing and changes in
matrix assembly during enamel development. Front. Physiol. 2017, 8, 790. [CrossRef] [PubMed]
127. Pandya, M.; Diekwisch, T.G.H. Enamel biomimetics—fiction or future of dentistry. Int. J. Oral Sci. 2019, 11, 8. [CrossRef]
128. Matsumoto, A.; Harada, H.; Saito, M.; Taniguchi, A. Induction of enamel matrix protein expression in an ameloblast cell line
co-cultured with a mesenchymal cell line in vitro. In Vitro Cell Dev. Biol. Anim. 2010, 47, 39–44. [CrossRef] [PubMed]
129. Honda, M.J.; Shinmura, Y.; Shinohara, Y. Enamel tissue engineering using subcultured enamel organ epithelial cells in combination
with dental pulp cells. Cells Tissues Organs 2009, 189, 261–267. [CrossRef] [PubMed]
130. Honda, M.J.; Shimodaira, T.; Ogaeri, T.; Shinohara, Y.; Hata, K.; Ueda, M. A novel culture system for porcine odontogenic
epithelial cells using a feeder layer. Arch. Oral Biol. 2006, 51, 282–290. [CrossRef] [PubMed]
131. Arakaki, M.; Ishikawa, M.; Nakamura, T.; Iwamoto, T.; Yamada, A.; Fukumoto, E.; Saito, M.; Otsu, K.; Harada, H.; Yamada, Y.;
et al. Role of epithelial-stem cell interactions during dental cell differentiation. J. Biol. Chem. 2012, 287, 10590–10601. [CrossRef]
[PubMed]
132. Kawano, S.; Morotomi, T.; Toyono, T.; Nakamura, N.; Uchida, T.; Ohishi, M.; Toyoshima, K.; Harada, H. Establishment of
dental epithelial cell line (HAT-7) and the cell differentiation depenDent. on notch signaling pathway. Connect. Tissue Res. 2002,
43, 409–412. [CrossRef] [PubMed]
133. Bori, E.; Guo, J.; Rácz, R.; Burghardt, B.; Földes, A.; Kerémi, B.; Harada, H.; Steward, M.C.; Besten, P.D.; Bronckers, A.L.J.J.; et al.
Evidence for bicarbonate secretion by ameloblasts in a novel cellular model. J. Dent. Res. 2016, 95, 588–596. [CrossRef] [PubMed]
134. Nakamura, T.; Chiba, Y.; Naruse, M.; Saito, K.; Harada, H.; Fukumoto, S. Globoside accelerates the differentiation of dental
epithelial cells into ameloblasts. Int. J. Oral Sci. 2016, 8, 205–212. [CrossRef] [PubMed]
135. Nakata, A.; Kameda, T.; Nagai, H.; Ikegami, K.; Duan, Y.; Terada, K.; Sugiyama, T. Establishment and characterization of a
spontaneously immortalized mouse ameloblast-lineage cell line. BioChem. Biophys. Res. Commun. 2003, 308, 834–839. [CrossRef]
136. Sidaly, R.; Landin, M.A.; Suo, Z.; Snead, M.L.; Lyngstadaas, S.P.; Reseland, J.E. Hypoxia increases the expression of enamel genes
and cytokines in an ameloblast-derived cell line. Eur. J. Oral Sci. 2015, 123, 335–340. [CrossRef]
137. Huang, Z.; Sargeant, T.D.; Hulvat, J.F.; Mata, A.; Bringas, P.; Koh, C.-Y.; Stupp, S.I.; Snead, M.L. Bioactive nanofibers instruct cells
to proliferate and differentiate during enamel regeneration. J. Bone Miner. Res. 2008, 23, 1995–2006. [CrossRef] [PubMed]
138. Sarkar, J.; Simanian, E.J.; Tuggy, S.Y.; Bartlett, J.D.; Snead, M.L.; Sugiyama, T.; Paine, M.L. Comparison of two mouse ameloblast-
like cell lines for enamel-specific gene expression. Front. Physiol. 2014, 5. [CrossRef] [PubMed]
Materials 2021, 14, 2558 32 of 35

139. Huang, Z.; Newcomb, C.J.; Zhou, Y.; Lei, Y.P.; Bringas, P., Jr.; Stupp, S.I.; Snead, M.L. The role of bioactive nanofibers in enamel
regeneration mediated through integrin signals acting upon C/EBP↵ and c-Jun. Biomaterials 2013, 34, 3303–3314. [CrossRef]
[PubMed]
140. Cai, J.; Zhang, Y.; Liu, P.; Chen, S.; Wu, X.; Sun, Y.; Li, A.; Huang, K.; Luo, R.; Wang, L.; et al. Generation of tooth-like structuRes.
from integration-free human urine induced pluripotent stem cells. Cell Regen. 2013, 2, 2–6. [CrossRef] [PubMed]
141. Liu, Y.; Jiang, M.; Hao, W.; Liu, W.; Tang, L.; Liu, H.; Jin, Y. Skin epithelial cells as possible substitutes for ameloblasts during
tooth regeneration. J. Tissue Eng. Regen. Med. 2012, 7, 934–943. [CrossRef] [PubMed]
142. Shinmura, Y.; Tsuchiya, S.; Hata, K.; Honda, M.J. Quiescent epithelial cell rests of Malassez can differentiate into ameloblast-like
cells. J. Cell Physiol. 2008, 217, 728–738. [CrossRef]
143. Wang, B.; Li, L.; Du, S.; Liu, C.; Lin, X.; Chen, Y.; Zhang, Y. Induction of human keratinocytes into enamel-secreting ameloblasts.
Dev. Biol. 2010, 344, 795–799. [CrossRef] [PubMed]
144. Chavez, M.G.; Hu, J.; Seidel, K.; Li, C.; Jheon, A.; Naveau, A.; Horst, O.; Klein, O.D. Isolation and culture of dental epithelial stem
cells from the adult mouse incisor. J. Vis. Exp. 2014, 87, 51266. [CrossRef] [PubMed]
145. Moussa, D.G.; Aparicio, C. Present and future of tissue engineering scaffolds for dentin-pulp complex regeneration. J. Tissue Eng.
Regen. Med. 2019, 13, 58–75. [CrossRef]
146. Sakai, V.T.; Zhang, Z.; Dong, Z.; Neiva, K.G.; Machado, M.A.A.M.; Shi, S.; Santos, S.S.; Nor, J.E. SHED differentiate into functional
odontoblasts and endothelium. J. Dent. Res. 2010, 89, 791–796. [CrossRef] [PubMed]
147. Huang, G.T.-J.; Yamaza, T.; Shea, L.D.; Djouad, F.; Kuhn, N.Z.; Tuan, R.S.; Shi, S. Stem/progenitor cell–mediated de novo
regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng. Part A 2010,
16, 605–615. [CrossRef]
148. Chen, Y.-J.; Zhao, Y.-H.; Zhao, Y.-J.; Liu, N.-X.; Lv, X.; Li, Q.; Chen, F.-M.; Zheng, M. Potential dental pulp revascularization and
odonto-/osteogenic capacity of a novel transplant combined with dental pulp stem cells and platelet-rich fibrin. Cell Tissue Res.
2015, 361, 439–455. [CrossRef] [PubMed]
149. Ruangsawasdi, N.; Zehnder, M.; Weber, F.E. Fibrin gel improves tissue ingrowth and cell differentiation in human immature
premolars implanted in rats. J. Endod. 2014, 40, 246–250. [CrossRef] [PubMed]
150. Iohara, K.; Imabayashi, K.; Ishizaka, R.; Watanabe, A.; Nabekura, J.; Ito, M.; Matsushita, K.; Nakamura, H.; Nakashima, M.
Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1. Tissue
Eng. Part A 2011, 17, 1911–1920. [CrossRef] [PubMed]
151. Kim, J.Y.; Xin, X.; Moioli, E.K.; Chung, J.; Lee, C.H.; Chen, M.; Fu, S.Y.; Koch, P.D.; Mao, J.J. Regeneration of dental-pulp-like
tissue by chemotaxis-induced cell homing. Tissue Eng. Part A 2010, 16, 3023–3031. [CrossRef] [PubMed]
152. Galler, K.M.; Hartgerink, J.D.; Cavender, A.C.; Schmalz, G.; D’Souza, R.N. A customized self-assembling peptide hydrogel for
dental pulp tissue engineering. Tissue Eng. Part A 2012, 18, 176–184. [CrossRef]
153. Rosa, V.; Zhang, Z.; Grande, R.H.M.; Nör, J.E. Dental pulp tissue engineering in full-length human root canals. J. Dent. Res. 2013,
92, 970–975. [CrossRef]
154. Galler, K.M.; Cavender, A.C.; Koeklue, U.; Suggs, L.J.; Schmalz, G.; D’Souza, R.N. Bioengineering of dental stem cells in a
PEGylated fibrin gel. Regen. Med. 2011, 6, 191–200. [CrossRef] [PubMed]
155. Simon, S.; Perard, M.; Zanini, M.; Smith, A.J.; Charpentier, E.; Djole, S.X.; Lumley, P.J. Should pulp chamber pulpotomy be seen as
a permanent treatment? Some preliminary thoughts. Int. Endod. J. 2012, 46, 79–87. [CrossRef]
156. Taha, N.A.; Khazali, M.A. Partial pulpotomy in mature permanent teeth with clinical signs indicative of irreversible pulpitis: A
randomized clinical trial. J. Endod. 2017, 43, 1417–1421. [CrossRef] [PubMed]
157. Qudeimat, M.A.; Alyahya, A.; Hasan, A.A.; Barrieshi-Nusair, K.M. Mineral trioxide aggregate pulpotomy for permanent molars
with clinical signs indicative of irreversible pulpitis: A preliminary study. Int. Endod. J. 2016, 50, 126–134. [CrossRef] [PubMed]
158. Mandakhbayar, N.; El-Fiqi, A.; Lee, J.-H.; Kim, H.-W. Evaluation of strontium-doped nanobioactive glass cement for dentin–pulp
complex regeneration therapy. ACS Biomater-Sci. Eng. 2019, 5, 6117–6126. [CrossRef] [PubMed]
159. Rad, R.M.; Atila, D.; Akgün, E.E.; Evis, Z.; Keskin, D.; Tezcaner, A. Evaluation of human dental pulp stem cells behavior on a
novel nanobiocomposite scaffold prepared for regenerative endodontics. Mater. Sci. Eng. C 2019, 100, 928–948.
160. Kontonasaki, E.; Bakopoulou, A.; Theocharidou, A.; Theodorou, G.S.; Papadopoulou, L.; Kantiranis, N.; Bousnaki, M.; Chatzichris-
tou, C.; Papachristou, E.; Paraskevopoulos, K.M.; et al. Effective cell growth potential of Mg-based bioceramic scaffolds towards
targeted dentin regeneration. Balk. J. Dent. Med. 2015, 19, 75–85. [CrossRef]
161. Soares, D.G.; Rosseto, H.L.; Basso, F.G.; Scheffel, D.S.; Hebling, J.; Costa, C.A.d.S. Chitosan-collagen biomembrane embedded
with calcium-aluminate enhances dentinogenic potential of pulp cells. Braz. Oral Res. 2016, 30, e54. [CrossRef] [PubMed]
162. Tran, H.L.B.; Doan, V.N. Human dental pulp stem cells cultured onto dentin derived scaffold can regenerate dentin-like tissue
in vivo. Cell Tissue Bank. 2015, 16, 559–568. [CrossRef] [PubMed]
163. Neves, V.C.M.; Babb, R.; Chandrasekaran, D.; Sharpe, P.T. Promotion of natural tooth repair by small molecule GSK3 antagonists.
Sci. Rep. 2017, 7, 39654. [CrossRef]
164. Cordeiro, M.M.; Dong, Z.; Kaneko, T.; Zhang, Z.; Miyazawa, M.; Shi, S.; Smith, A.J.; Nor, J.E. Dental pulp tissue engineering with
stem cells from exfoliated deciduous teeth. J. Endod. 2008, 34, 962–969. [CrossRef]
165. Ouchi, T.; Nakagawa, T. Mesenchymal stem cell-based tissue regeneration therapies for periodontitis. Regen. Ther. 2020, 14, 72–78.
[CrossRef]
Materials 2021, 14, 2558 33 of 35

166. Yang, Y.; Rossi, F.M.V.; Putnins, E.E. Periodontal regeneration using engineered bone marrow mesenchymal stromal cells.
Biomaterials 2010, 31, 8574–8582. [CrossRef] [PubMed]
167. Gay, I.; Chen, S.; MacDougall, M. Isolation and characterization of multipotent human periodontal ligament stem cells. Orthod.
Craniofac. Res. 2007, 10, 149–160. [CrossRef]
168. Han, C.; Yang, Z.; Zhou, W.; Jin, F.; Song, Y.; Wang, Y.; Huo, N.; Chen, L.; Qian, H.; Hou, R.; et al. Periapical follicle stem
cell: A promising candidate for cementum/periodontal ligament regeneration and bio-root engineering. Stem Cells Dev. 2010,
19, 1405–1415. [CrossRef] [PubMed]
169. Nuñez, J.; Sanz-Blasco, S.; Vignoletti, F.; Muñoz, F.; Arzate, H.; Villalobos, C.; Nuñez, L.; Caffesse, R.G.; Sanz, M. Periodontal
regeneration following implantation of cementum and periodontal ligament-derived cells. J. Periodont. Res. 2011, 47, 33–44.
[CrossRef] [PubMed]
170. Arzate, H.; Zeichner-David, M.; Mercado-Celis, G. Cementum proteins: Role in cementogenesis, biomineralization, periodontium
formation and regeneration. Periodontol. 2000 2014, 67, 211–233. [CrossRef] [PubMed]
171. Hoz, L.; Romo, E.; Zeichner-David, M.; Sanz, M.; Nuñez, J.; Gaitán, L.; Mercado, G.; Arzate, H. Cementum protein 1 (CEMP1)
induces differentiation by human periodontal ligament cells under three-dimensional culture conditions. Cell Biol. Int. 2011,
36, 129–136. [CrossRef]
172. Bosshardt, D.D.; Schroeder, H.E. Cementogenesis reviewed: A comparison between human premolars and rodent molars. Anat.
Rec. 1996, 245, 267–292. [CrossRef]
173. Zhu, W.; Liang, M. Periodontal ligament stem cells: Current status, concerns, and future prospects. Stem Cells Int. 2015, 2015, 1–11.
[CrossRef]
174. Yang, B.; Chen, G.; Li, J.; Zou, Q.; Xie, D.; Chen, Y.; Wang, H.; Zheng, X.; Long, J.; Tang, W.; et al. Tooth root regeneration using
dental follicle cell sheets in combination with a dentin matrix—Based scaffold. Biomaterials 2012, 33, 2449–2461. [CrossRef]
175. Ivanovski, S.; Vaquette, C.; Gronthos, S.; Hutmacher, D.W.; Bartold, P.M. Multiphasic scaffolds for periodontal tissue engineering.
J. Dent. Res. 2014, 93, 1212–1221. [CrossRef] [PubMed]
176. Jeon, J.E.; Vaquette, C.; Klein, T.J.; Hutmacher, D.W. Perspectives in multiphasic osteochondral tissue engineering. Anat. Rec.
2013, 297, 26–35. [CrossRef] [PubMed]
177. Akizuki, T.; Oda, S.; Komaki, M.; Tsuchioka, H.; Kawakatsu, N.; Kikuchi, A.; Yamato, M.; Okano, T.; Ishikawa, I. Application of
periodontal ligament cell sheet for periodontal regeneration: A pilot study in beagle dogs. J. Periodontal Res. 2005, 40, 245–251.
[CrossRef] [PubMed]
178. Gomez Flores, M.; Hasegawa, M.; Yamato, M.; Takagi, R.; Okano, T.; Ishikawa, I. Cementum-periodontal ligament complex
regeneration using the cell sheet technique. J. Periodontal Res. 2008, 43, 364–371. [CrossRef] [PubMed]
179. Hasegawa, M.; Yamato, M.; Kikuchi, A.; Okano, T.; Ishikawa, I. Human periodontal ligament cell sheets can regenerate periodontal
ligament tissue in an athymic rat model. Tissue Eng. 2005, 11, 469–478. [CrossRef] [PubMed]
180. Ishikawa, I.; Iwata, T.; Washio, K.; Okano, T.; Nagasawa, T.; Iwasaki, K.; Ando, T. Cell sheet engineering and oTher. novel
cell-based approaches to periodontal regeneration. Periodontol. 2000 2009, 51, 220–238. [CrossRef] [PubMed]
181. Iwata, T.; Yamato, M.; Tsuchioka, H.; Takagi, R.; Mukobata, S.; Washio, K.; Okano, T.; Ishikawa, I. Periodontal regeneration with
multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials 2009, 30, 2716–2723. [CrossRef] [PubMed]
182. Iwata, T.; Yamato, M.; Zhang, Z.; Mukobata, S.; Washio, K.; Ando, T.; Feijen, J.; Ishikawa, I. Validation of human periodontal
ligament-derived cells as a reliable source for cytotherapeutic use. J. Clin. Periodontol. 2010, 37, 1088–1099. [CrossRef]
183. Flores, M.G.; Yashiro, R.; Washio, K.; Yamato, M.; Okano, T.; Ishikawa, I. Periodontal ligament cell sheet promotes periodontal
regeneration in athymic rats. J. Clin. Periodontol. 2008, 35, 1066–1072. [CrossRef]
184. Vaquette, C.; Fan, W.; Xiao, Y.; Hamlet, S.; Hutmacher, D.W.; Ivanovski, S. A biphasic scaffold design combined with cell sheet
technology for simultaneous regeneration of alveolar bone/periodontal ligament complex. Biomaterials 2012, 33, 5560–5573.
[CrossRef]
185. Anusaksathien, O.; Jin, Q.; Zhao, M.; Somerman, M.J.; Giannobile, W.V. Effect of sustained gene delivery of platelet-derived
growth factor or its antagonist (PDGF-1308) on tissue-engineered cementum. J. Periodontol. 2004, 75, 429–440. [CrossRef]
186. Jin, Q.-M.; Zhao, M.; Webb, S.A.; Berry, J.E.; Somerman, M.J.; Giannobile, W.V. Cementum engineering with three-dimensional
polymer scaffolds. J. BioMed. Mater. Res. A 2003, 67, 54–60. [CrossRef] [PubMed]
187. Park, C.H.; Rios, H.F.; Jin, Q.; Bland, M.E.; Flanagan, C.L.; Hollister, S.J.; Giannobile, W.V. Biomimetic hybrid scaffolds for
engineering human tooth-ligament interfaces. Biomaterials 2010, 31, 5945–5952. [CrossRef] [PubMed]
188. Park, C.H.; Rios, H.F.; Jin, Q.; Sugai, J.V.; Padial-Molina, M.; Taut, A.D.; Flanagan, C.L.; Hollister, S.J.; Giannobile, W.V. Tissue
engineering bone-ligament complexes using fiber-guiding scaffolds. Biomaterials 2012, 33, 137–145. [CrossRef] [PubMed]
189. Park, C.H.; Rios, H.F.; Taut, A.D.; Padial-Molina, M.; Flanagan, C.L.; Pilipchuk, S.P.; Hollister, S.J.; Giannobile, W.V. Image-based,
fiber guiding scaffolds: A platform for regenerating tissue interfaces. Tissue Eng. Part C Methods 2014, 20, 533–542. [CrossRef]
[PubMed]
190. Lee, C.H.; Hajibandeh, J.; Suzuki, T.; Fan, A.; Shang, P.; Mao, J.J. Three-dimensional printed multiphase scaffolds for regeneration
of periodontium complex. Tissue Eng. Part C Methods A 2014, 20, 1342–1351. [CrossRef]
191. Cho, H.; Tarafder, S.; Fogge, M.; Kao, K.; Lee, C.H. Periodontal ligament stem/progenitor cells with protein-releasing scaffolds
for cementum formation and integration on dentin surface. Connect. Tissue Res. 2016, 57, 488–495. [CrossRef]
Materials 2021, 14, 2558 34 of 35

192. Mao, L.X.; Liu, J.; Zhao, J.; Xia, L.; Jiang, L.; Wang, X.; Lin, K.; Fang, B. Effect of micro-nano-hybrid structured hydroxyapatite
bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway.
Int. J. Nanomed. 2015, 10, 7031. [CrossRef] [PubMed]
193. Chronopoulou, L.; Cacciotti, I.; Amalfitano, A.; Di Nitto, A.; D’Arienzo, V.; Nocca, G.; Paloci, C. Biosynthesis of innovative
calcium phosphate/hydrogel composites: Physicochemical and biological characterisation. Nanotechnology 2020, 32, 095102.
[CrossRef]
194. Takayama, S.; Murakami, S.; Shimabukuro, Y.; Kitamura, M.; Okada, H. Periodontal Regeneration by FGF-2 (bFGF) in Primate
Models. J. Dent. Res. 2001, 80, 2075–2079. [CrossRef]
195. Murakami, S.; Takayama, S.; Kitamura, M.; Shimabukuro, Y.; Yanagi, K.; Ikezawa, K.; Okada, H. Recombinant human basic
fibroblast growth factor (bFGF) stimulates periodontal regeneration in class II furcation defects created in beagle dogs. J.
Periodontal Res. 2003, 38, 97–103. [CrossRef] [PubMed]
196. Sowmya, S.; Mony, U.; Jayachandran, P.; Reshma, S.; Kumar, R.A.; Arzate, H.; Nair, S.V.; Jayakumar, R. Tri-layered nanocomposite
hydrogel scaffold for the concurrent regeneration of cementum, periodontal ligament, and alveolar bone. Adv. Healthc. Mater.
2017, 6, 1601251. [CrossRef] [PubMed]
197. Monteiro, N.; Yelick, P.C. Advances and perspectives in tooth tissue engineering. J. Tissue Eng. Regen. Med. 2016, 11, 2443–2461.
[CrossRef] [PubMed]
198. Li, L.; Tang, Q.; Wang, A.; Chen, Y. Regrowing a tooth: In vitro and in vivo approaches. Curr. Opin. Cell Biol. 2019, 61, 126–131.
[CrossRef] [PubMed]
199. Ramanathan, A.; Srijaya, T.C.; Sukumaran, P.; Zain, R.B.; Abu Kasim, N.H. Homeobox genes and tooth development: Under-
standing the biological pathways and applications in regenerative dental science. Arch. Oral Biol. 2018, 85, 23–39. [CrossRef]
[PubMed]
200. Nakao, K.; Morita, R.; Saji, Y.; Ishida, K.; Tomita, Y.; Ogawa, M.; Saitoh, M.; Tomooka, Y.; Tsuji, T. The development of a
bioengineered organ germ method. Nat. Methods 2007, 4, 227–230. [CrossRef] [PubMed]
201. Lai, W.-F.; Lee, J.-M.; Jung, H.-S. Molecular and engineering approaches to regenerate and repair teeth in mammals. Cell. Mol. Life
Sci. 2013, 71, 1691–1701. [CrossRef] [PubMed]
202. Li, G.; Zhang, T.; Li, M.; Fu, N.; Fu, Y.; Ba, K.; Deng, S.; Jiang, Y.; Hu, J.; Peng, Q.; et al. Electrospun fibers for dental and
craniofacial applications. Curr. Stem Cell Res. T 2014, 9, 187–195. [CrossRef] [PubMed]
203. Takahashi, K.; Kiso, H.; Saito, K.; Togo, Y.; Tsukamoto, H.; Huang, B.; Bessho, K. Feasibility of gene therapy for tooth regeneration
by stimulation of a third dentition. In Gene Therapy—Tools and Potential Applications; InTech: London, UK, 2013.
204. Hu, X.; Lee, J.-W.; Zheng, X.; Zhang, J.; Lin, X.; Song, Y.; Wang, B.; Hu, X.; Chang, H.-H.; Chen, Y.; et al. Efficient induction of
functional ameloblasts from human keratinocyte stem cells. Stem Cell Res. Ther. 2018, 9, 126. [CrossRef]
205. Lechguer, A.N.; Kuchler-Bopp, S.; Hu, B.; Haïkel, Y.; Lesot, H. Vascularization of engineered teeth. J. Dent. Res. 2008, 87, 1138–1143.
[CrossRef]
206. Lechguer, A.N.; Couble, M.L.; Labert, N.; Kuchler-Bopp, S.; Keller, L.; Magloire, H.; Bleicher, F.; Lesot, H. Cell differentiation and
matrix organization in engineered teeth. J. Dent. Res. 2011, 90, 583–589. [CrossRef]
207. Hu, B.; Nadiri, A.; Bopp-Küchler, S.; Perrin-Schmitt, F.; Lesot, H. Dental epithelial histomorphogenesis in vitro. J. Dent. Res. 2005,
84, 521–525. [CrossRef] [PubMed]
208. Hu, B.; Nadiri, A.; Kuchler-Bopp, S.; Perrin-Schmitt, F.; Peters, H.; Lesot, H. Tissue engineering of tooth crown, root, and
periodontium. Tissue Eng. 2006, 12, 2069–2075. [CrossRef] [PubMed]
209. Honda, M.J.; Fong, H.; Iwatsuki, S.; Sumita, Y.; Sarikaya, M. Tooth-forming potential in embryonic and postnatal tooth bud cells.
Med. Mol. Morphol. 2008, 41, 183–192. [CrossRef] [PubMed]
210. Donoghue, P.C.J.; Rücklin, M. The ins and outs of the evolutionary origin of teeth. Evol. Dev. 2014, 18, 19–30. [CrossRef] [PubMed]
211. Abduweli, D.; Baba, O.; Tabata, M.J.; Higuchi, K.; Mitani, H.; Takano, Y. Tooth replacement and putative odontogenic stem cell
niches in pharyngeal dentition of medaka (Oryziaslatipes). Microscopy 2014, 63, 141–153. [CrossRef]
212. Jernvall, J.; Thesleff, I. Tooth shape formation and tooth renewal: Evolving with the same signals. Development 2012, 139, 3487–3497.
[CrossRef] [PubMed]
213. Juuri, E.; Jussila, M.; Seidel, K.; Holmes, S.; Wu, P.; Richman, J.; Heikinheimo, K.; Chuong, C.-M.; Arnold, K.; Hochedlinger, K.;
et al. Sox2 marks epithelial competence to generate teeth in mammals and reptiles. Development 2013, 140, 1424–1432. [CrossRef]
214. Wu, P.; Wu, X.; Jiang, T.-X.; Elsey, R.M.; Temple, B.L.; Divers, S.J.; Glenn, T.C.; Yuan, K.; Chen, M.H.; Widelitz, R.B.; et al.
Specialized stem cell niche enables repetitive renewal of alligator teeth. Proc. Natl. Acad. Sci. USA 2013, 110, E2009–E2018.
[CrossRef] [PubMed]
215. Hu, B.; Nadiri, A.; Bopp-Kuchler, S.; Perrin-Schmitt, F.; Wang, S.; Lesot, H. Dental epithelial histo-morphogenesis in the mouse:
Positional information versus cell history. Arch. Oral Biol. 2005, 50, 131–136. [CrossRef] [PubMed]
216. Ikeda, E.; Tsuji, T. Growing bioengineered teeth from single cells: Potential for dental regenerative medicine. Expert Opin. Biol.
Ther. 2008, 8, 735–744. [CrossRef] [PubMed]
217. Ishida, K.; Murofushi, M.; Nakao, K.; Morita, R.; Ogawa, M.; Tsuji, T. The regulation of tooth morphogenesis is associated with
epithelial cell proliferation and the expression of Sonic hedgehog through epithelial–mesenchymal interactions. BioChem. Biophys.
Res. Commun. 2011, 405, 455. [CrossRef] [PubMed]
Materials 2021, 14, 2558 35 of 35

218. Ikeda, E.; Morita, R.; Nakao, K.; Ishida, K.; Nakamura, T.; Takano-Yamamoto, T.; Ogawa, M.; Mizuno, M.; Kasugai, S.; Tsuji, T. Fully
functional bioengineered tooth replacement as an organ replacement therapy. Proc. Natl. Acad. Sci. USA 2009, 106, 13475–13480.
[CrossRef]
219. Hu, X.; Lin, C.; Shen, B.; Ruan, N.; Guan, Z.; Zhang, Y. Conserved odontogenic potential in embryonic dental tissues. J. Dent. Res.
2014, 93, 490–495. [CrossRef] [PubMed]
220. Egusa, H.; Sonoyama, W.; Nishimura, M.; Atsuta, I.; Akiyama, K. Stem cells in dentistry—Part II: Clinical applications. J.
Prosthodont. Res. 2012, 56, 229–248. [CrossRef]
221. Ohazama, A.; Modino, S.A.C.; Miletich, I.; Sharpe, P.T. Stem-cell-based tissue engineering of murine teeth. J. Dent. Res. 2004,
83, 518–522. [CrossRef]
222. Modino, S.A.C.; Sharpe, P.T. Tissue engineering of teeth using adult stem cells. Arch. Oral Biol. 2005, 50, 255–258. [CrossRef]
[PubMed]
223. Kaneko, T.; Sone, P.P.; Zaw, S.Y.M.; Sueyama, Y.; Zaw, Z.C.T.; Okada, Y.; Murano, H.; Gu, B.; Okiji, T. In vivo fate of bone marrow
mesenchymal stem cells implanted into rat pulpotomized molars. Stem Cell Res. 2019, 38, 101457. [CrossRef] [PubMed]
224. Wen, Y.; Wang, F.; Zhang, W.; Li, Y.; Yu, M.; Nan, X.; Chen, L.; Yue, X.; Xu, X.; Pei, X. Application of induced pluripotent stem cells
in generation of a tissue-engineered tooth-like. Tissue Eng. Part A 2012, 18, 1677–1685. [CrossRef]
225. Otsu, K.; Kishigami, R.; Oikawa-Sasaki, A.; Fukumoto, S.; Yamada, A.; Fujiwara, N.; Ishizeki, K.; Harada, H. Differentiation of
induced pluripotent stem cells into dental mesenchymal cells. Stem Cells Dev. 2012, 21, 1156–1164. [CrossRef]
226. Liu, L.; Liu, Y.-F.; Zhang, J.; Duan, Y.-Z.; Jin, Y. Ameloblasts serum-free conditioned medium: Bone morphogenic protein
4-induced odontogenic differentiation of mouse induced pluripotent stem cells. J. Tissue Eng. Regen. Med. 2013, 10, 466–474.
[CrossRef]
227. Smith, E.E.; Zhang, W.; Schiele, N.R.; Khademhosseini, A.; Kuo, C.K.; Yelick, P.C. Developing a biomimetic tooth bud model. J.
Tissue Eng. Regen. Med. 2017, 11, 3326–3336. [CrossRef]
228. Khayat, A.; Monteiro, N.; Smith, E.E.; Pagni, S.; Zhang, W.; Khademhosseini, A.; Yelik, P.C. GelMA-encapsulated hDPSCs and
HUVECs for dental pulp regeneration. J. Dent. Res. 2016, 96, 192–199. [CrossRef]
229. Smith, E.E.; Yelick, P.C. Bioengineering tooth bud constructs using GelMA hydrogel. Methods Mol. Biol. 2019, 1922, 139–150.
[PubMed]
230. Monteiro, N.; Smith, E.E.; Angstadt, S.; Zhang, W.; Khademhosseini, A.; Yelick, P.C. Dental cell sheet biomimetic tooth bud model.
Biomaterials 2016, 106, 167–179. [CrossRef] [PubMed]
231. Smith, E.E.; Angstadt, S.; Monteiro, N.; Zhang, W.; Khademhosseini, A.; Yelick, P.C. Bioengineered tooth buds exhibit featuRes. of
natural tooth buds. J. Dent. Res. 2018, 97, 1144–1151. [CrossRef] [PubMed]
232. Iwatsuki, S.; Honda, M.J.; Harada, H.; Ueda, M. Cell proliferation in teeth reconstructed from dispersed cells of embryonic tooth
germs in a three-dimensional scaffold. Eur. J. Oral Sci. 2006, 114, 310–317. [CrossRef]
233. Chen, G.; Chen, J.; Yang, B.; Li, L.; Luo, X.; Zhang, X.; Feng, L.; Jiang, Z.; Yu, M.; Guo, W.; et al. Combination of aligned
PLGA/gelatin electrospun sheets, native dental pulp extracellular matrix and treated dentin matrix as substrates for tooth root
regeneration. Biomaterials 2015, 52, 56–70. [CrossRef]
234. Xu, W.P.; Zhang, W.; Asrican, R.; Kim, H.J.; Kaplan, D.L.; Yelick, P.C. Accurately shaped tooth bud cell-derived mineralized tissue
formation on silk scaffolds. Tissue Eng. Part A 2008, 14, 549–557. [CrossRef]
235. Traphagen, S.B.; Fourligas, N.; Xylas, J.F.; Sengupta, S.; Kaplan, D.L.; Georgakoudi, I.; Yelik, P.C. Characterization of natural,
decellularized and reseeded porcine tooth bud matrices. Biomaterials 2012, 33, 5287–5296. [CrossRef]
236. Zhang, W.; Vazquez, B.; Oreadi, D.; Yelick, P.C. Decellularized tooth bud scaffolds for tooth regeneration. J. Dent. Res. 2017,
96, 516–523. [CrossRef]
237. Sumita, Y.; Honda, M.J.; Ohara, T.; Tsuchiya, S.; Sagara, H.; Kagami, H.; Ueda, M. Performance of collagen sponge as a 3D scaffold
for tooth-tissue engineering. Biomaterials 2006, 27, 3238–3248. [CrossRef] [PubMed]
238. Song, J.S.; Takimoto, K.; Jeon, M.; Vadakekalam, J.; Ruparel, N.B.; Diogenes, A. Decellularized human dental pulp as a scaffold for
regenerative endodontics. J. Dent. Res. 2017, 96, 640–646. [CrossRef] [PubMed]